journal of materials chemistry b - nam-trung nguyen · 2019. 11. 7. · his ournal is ' he...

REVIEW ARTICLE Muhammad J. A. Shiddiky et al . Advanced liquid biopsy technologies for circulating biomarker detection

Journal of Materials Chemistry BMaterials for biology and medicine

ISSN 2050-750X

rsc.li/materials-b

Volume 7 Number 43 21 November 2019 Pages 6647–6870

6670 | J. Mater. Chem. B, 2019, 7, 6670--6704 This journal is©The Royal Society of Chemistry 2019

Cite this: J.Mater. Chem. B, 2019,

7, 6670

Advanced liquid biopsy technologies forcirculating biomarker detection

Narshone Soda, ab Bernd H. A. Rehm, c Prashant Sonar, d

Nam-Trung Nguyen b and Muhammad J. A. Shiddiky *ab

Liquid biopsy is a new diagnostic concept that provides important information for monitoring and

identifying tumor genomes in body fluid samples. Detection of tumor origin biomolecules like circulating

tumor cells (CTCs), circulating tumor specific nucleic acids (circulating tumor DNA (ctDNA), circulating

tumor RNA (ctRNA), microRNAs (miRNAs), long non-coding RNAs (lnRNAs)), exosomes, autoantibodies in

blood, saliva, stool, urine, etc. enables cancer screening, early stage diagnosis and evaluation of therapy

response through minimally invasive means. From reliance on painful and hazardous tissue biopsies or

imaging depending on sophisticated equipment, cancer management schemes are witnessing a rapid

evolution towards minimally invasive yet highly sensitive liquid biopsy-based tools. Clinical application of

liquid biopsy is already paving the way for precision theranostics and personalized medicine. This is

achieved especially by enabling repeated sampling, which in turn provides a more comprehensive

molecular profile of tumors. On the other hand, integration with novel miniaturized platforms, engineered

nanomaterials, as well as electrochemical detection has led to the development of low-cost and simple

platforms suited for point-of-care applications. Herein, we provide a comprehensive overview of the

biogenesis, significance and potential role of four widely known biomarkers (CTCs, ctDNA, miRNA and

exosomes) in cancer diagnostics and therapeutics. Furthermore, we provide a detailed discussion of the

inherent biological and technical challenges associated with currently available methods and the

possible pathways to overcome these challenges. The recent advances in the application of a wide

range of nanomaterials in detecting these biomarkers are also highlighted.

1. Introduction

Early detection of circulating biomarkers (CBs) in accessiblebody fluids such as blood or urine has the potential to improvesurvival for individuals burdened with cancer. It also decreases thecost of treatment by ameliorating the prescription of ineffectivetherapies. The four most common CBs with excellent diagnostic,prognostic and therapeutic potential are circulating tumour cells(CTCs),1 circulating tumor specific nucleic acids (ctDNA, ctRNA,miRNAs, lnRNAs),2,3 extracellular vesicles4 (exosomes, apoptopicbodies, etc.), and autoantibodies.5 By capturing these CBs inclinical samples, physicians can essentially perform ‘liquid biopsy’.This is a promising non-invasive or minimally invasive diagnostictool for early diagnosis of cancer that provides real-time

information on tumor evolution and therapeutic response incontrast to conventional tissue biopsy.6 The conventionaltumor tissue biopsies are severely prone to sampling bias –since repeated sample collection is not feasible they onlyprovide a glimpse of tumor heterogeneity.7 Thus, liquidbiopsies have emerged as a disparate group of technologiesthat seek to expand the scope and application of body fluid-based cancer diagnosis. Liquid biopsy also provides insightsinto tumor biology and potentially can differentiate metastaticand indolent cancer. Thus, an opportunity to identify reliableCBs mirroring tumor behavior, via fully or minimally non-invasive liquid biopsy, represents a great paradigm shift inpersonalized clinical care.

Owing to the recent advances in the development of highlyspecific gene-amplification and sequencing technologies liquidbiopsies can access more biomarkers relevant to cancer. Thus,the molecular profile of liquid biopsies is a promising fieldfor cancer biomarker discovery. This is due to the relativelylow invasive techniques used to collect the material and thepossibility of obtaining many samples from the same individualsat different times. For many years, CTCs, exosomes, ctDNA andmicroRNAs have been regarded as major biomarkers for cancer

a School of Environment and Science, Griffith University, Nathan Campus,

QLD 4111, Australia. E-mail: [email protected] Queensland Micro- and Nanotechnology Centre (QMNC), Griffith University,

Nathan Campus, QLD 4111, Australiac Centre for Cell Factories and Biopolymers (CCFB), Griffith Institute for Drug

Discovery (GRIDD), Griffith University, Nathan, QLD 4111, Australiad School of Chemistry, Physics and Mechanical Engineering, Molecular Design and

Synthesis, Queensland University of Technology (QUT), Brisbane, Australia

Received 18th July 2019,Accepted 25th September 2019

DOI: 10.1039/c9tb01490j

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Journal ofMaterials Chemistry B

REVIEW

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http://orcid.org/0000-0003-3908-8903

http://orcid.org/0000-0002-1119-4897

http://orcid.org/0000-0003-3626-5361

http://orcid.org/0000-0003-4526-4109

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detection.6 These biomarkers are released from tumor sites intoperipheral fluids; hence they can be detected and analyzed.The large amount of cancer cells in circulation is driven by acontinuous discharge of tumor-derived nucleic acids, viableCTCs and exosomes into the circulation. In brief, the ability toisolate, characterize and detect these CBs using non-invasiveapproaches represents a new diagnostic tool that would enableclinical experts to analyze the evolution of tumor multiple times(Fig. 1).

The need for non-invasive molecular profiling tools in recentyears has grown substantially due to the increasing need andbetter understanding of genomic alterations and personalizedtreatment options. The integration and utilization of CBsin routine clinical settings is of utmost importance. Hence,numerous validation studies are required to provide substan-tial evidence of the efficiency and reliability of the markers forthe clinical utility of the developed tests. In the past few years,many technologies have been developed for the detection ofCBs in peripheral blood using different platforms.2,4,8,9 In spiteof this technological advancement, the lack of standardizationand variability in methodologies for CB assessment contributeto dynamic limitations for integrating these technologies inclinical settings. A fundamental prerequisite in the develop-ment of CB-based diagnostics is the ability to measure CBsfrom body fluid (serum, plasma, etc.) with adequate precisionand sensitivity. The precise quantification of CBs has beengreatly influenced by many limitations such as pre-analyticvariations, technical problems in qRT-PCR and data analysis,and normalization.10

The integration of liquid biopsies with novel miniaturizedplatforms, engineered nanomaterials, and electrochemical detectionhas led to the development of low-cost and simple platformssuited for point-of-care applications. Recently, several reviewshave been published on the application of CTCs, ctDNA, miRNA,and exosomes as cancer biomarkers,11–13 as well as on theirdetection strategies.2,14,15 Herein, we provide a comprehensivereview of the literature published in recent years on the back-ground of the biology of CTCs, ctDNA, miRNA and exosomes asbiomarkers. A detailed discussion of the inherent technicalchallenges together with possible solutions to overcome thesechallenges is provided. We also provide a critical commentary onthe performance attributes of various newly developed materialsand technologies. This will assist researchers in recognizingthe shortcomings and limitations of current platforms, a betterunderstanding of which can help in identifying possible futureavenues of research.

2. Biogenesis of CTCs, ctDNA, miRNAand exosomesCTCs

The earliest detection of CTCs in peripheral blood was reportedby Australian physician Thomas Ashworth using a microscopein 1860.16 Thereafter, a theory was proposed based on theinfiltration of tumor cells into the vessel wall and bloodstream.In particular, the majority of CTCs were found to be accidentalcells in circulation driven passively or actively by external forces

Fig. 1 Schematic representation of the circulating biomarkers, functions, and detection technologies in molecular diagnosis.

Review Journal of Materials Chemistry B


such as tumor growth and mechanical stress during surgicaloperation.17 The formation of metastatic lesions during cancerprogression was initially thought to occur in the later stages.However recent reports have demonstrated that CTCs infiltratethe circulation at an early stage and spread to potential metastaticregions as single cells or clusters.18,19 Numerous metastasisstudies conducted in the 20th century indicated that clusters oraggregates of tumor cells contain higher metastatic potential thansingle cells.20–22 A direct correlation of size, concentration andnumber of CTC aggregates with their metastatic potential wasestablished particularly in animal studies showing the capabilityof CTC aggregates in traversing pulmonary circulation in smallanimals.20,23 The analysis of CTCs is therefore centered on theevaluation of mechanisms of cancer metastasis. During meta-stasis initiation, the tumor cells in the primary site proliferateresulting in the attraction of blood vessels to provide oxygenand nutrients for tumor growth. Epithelial–mesenchymal tran-sition (EMT) has been linked to metastasis. However, itsinvolvement still remains unclear as to whether it occurs andto which degree it contributes to metastasis. Recent evidencesuggests that patients with metastatic lesions are more suscep-tible to have CTCs that can be easily isolated but they are usuallyvery low concentrated, usually 1–10 CTCs per mL of whole blood24

which also contains about 7 � 106 and 5 � 109 white blood cellsand red blood cells respectively.25 Therefore, technologies thatcan effectively isolate a single CTC from the backgroundof several blood components are essential. While such levelsof sensitivity are difficult to achieve, a considerable number ofnovel technologies have been developed to efficiently isolate,quantify and characterise rare CTCs.

ctDNA

Circulating tumor DNA was first reported by Mandal and Metailsin 1948.26 CtDNA is a fraction of cell-free DNA that emanatesfrom tumor cells and is discharged into the circulation. Cell-freeDNA comprises short nucleic acid fragments (B166 bp) foundin almost all body fluids, including plasma, and is likelyderived from apoptotic cells.27,28 cfDNA is involved in differentphysiological and pathological processes such as coagulation,immunity, aging, and cancer. In cancer patients, the largenumber of tumors leads to the release of circulating tumorDNA which may carry similar genetic alterations and mutationsas the primary tumor. Thus, the analysis of ctDNA could enablecancer diagnosis and prognosis through non-invasive means.Under normal physiological conditions, infiltrated phagocytesrelease necrotic and apoptotic cell debris from the tissue. Theincreased cellular turnover also results in a rapid increase of celldebris. Therefore, the removal of biological molecules in necro-tic and apoptotic cell debris into the circulation, includingctDNA, is substantially greater than in normal conditions. There-fore, ctDNA is considered as a potential biomarker for diagnosis,prognosis and patient-specific therapy.

RNA

Biogenesis of RNA in the nucleus and its function in generegulation and protein synthesis are greatly influenced by

several controlled pathways based on multiple enzymes andcellular factors.29–31 Circulating miRNAs represent short RNAmolecules with an average length of 22 nucleotides that play animportant role in regulating gene expression through bindingto target miRNA. miRNA synthesis involves the conversion ofmiRNA genes to hairpin-structured primary miRNA either byRNA polymerase II or via the transcription process.8 Followingthis conversion transcription miRNAs are split and processedby a distinctive microprocessor complex. This contains RNAbinding protein DGCR8 and ribonuclease enzyme Drosha.Subsequently, miRNAs are transferred to the cytoplasm andfurther divided by an enzyme called Dicer to form miRNAduplexes. Mature miRNAs are then produced after strandseparation of the duplexes and begin to assemble variousenzymes and proteins to produce RNA induced silencing com-plexes. The regulatory and inhibitory actions exerted by miRNAare driven by RNA silencing complex-induced degradation andpost-translational inhibition (Fig. 2).32,33

Exosomes

The discovery of exosomes was first reported three decades agoby two independent research groups that described the trans-ferring receptors in reticulocytes to be related to small-sizedvesicles approximately 50 nm in diameter. The specific roles ofthese vesicles were not known until later in 1996 when it wasestablished that exosomes are discharged by B immune cellsand can activate CD4+ T-cell clones in an antigen-specificmanner.4,35 The term exosome was then formulated to describevesicles with a nanosized diameter ranging between 30 and150 nm shed into the extracellular environment once there isfusion between endosomes and cellular membranes.36 Follow-ing this discovery, several extracellular vesicles have beenidentified and classified according to their biogenesis, functionand cellular genesis.37,38 Various mechanisms are involved inbiogenesis of exosomes, which also allow better classificationof the protein and RNA cargo to produce exosomes withdistinctive biochemical compositions. Exosomes exist in allbody fluids including urine and plasma and are generated bymost normal and pathological cells. The origin of exosomesfrom the multivesicular endosome (MVE) starts with the inwardinvagination of the cellular plasma membrane which generatesendosomes. The endosome membrane undergoes a series ofinward invaginations, which transform the early endosome tothe mature late endosome, resulting in the generation ofmultiple intraluminal vesicles. The multivesicular body (MVB)formed at this point consists of numerous vesicles with eachcontaining cytosol with different proteins and nucleic acids in asmall unit. Exosomes are therefore secreted into the extravesi-cular space when the MVB fuses with the plasma membrane.Exosomes can be secreted via the trans-Golgi network orinducible pathways. Various proteins from the Rab family suchas Rab27a and Rab27b function essentially as regulators ofexosome secretion.39 Previous studies have also shown that theactivation of tumor suppressor protein, p53, activates andincreases the exosome secretion rate by regulating the tran-scription of several genes including TSAP6 and CHMP4C.40

Journal of Materials Chemistry B Review


3. Diagnostic, prognostic andtherapeutic use of CBs3.1 CBs as diagnostic biomarkers

CtDNA. Early diagnosis of cancer is vital as it providesguidance towards effective therapeutic interventions andsignificant improvement in patient survival.41 Over the pastfew years, detection of ctDNA by non-invasive means has madeexceptional contribution towards the pursuit of potent andcredible biomarkers. Predictive biomarkers play a pivotal rolein guiding treatment decision. Patients are identified anddivided into subgroups depending on their responses to specificcancer therapies, or other alternative treatment procedures.Detection of specific cancer epigenetic aberrations using ctDNA,such as promoter hypomethylation, may aid to provide vitalinformation to apprehend tumor biology and analysis in clinicalenvironments. As a potential predictive biomarker, O6-methyl-guanine-methyl-transferase (MGMT) promoter methylation forctDNA can be measured in glioblastoma multiforme (GBM)patients. The measurement of MGMT methylation in plasmactDNA using bisulphite-pyrosequencing and methyl-BEAMingstrategies showed higher response of the MGMT methylationstatus in metastatic colorectal cancer. This provided betterprognosis of treatment response and improvement inprogression-free survival.42 Assimilation of ctDNA in potentialclinical assays can aid in the identification and classification ofpredictive biomarkers based on their response towards therapyusing key somatic mutations. Earlier studies reported selective

detection of circulating mutant KRAS in non-small cell lungcancer patients by using restriction fragment-length polymorphismand polymerase chain reaction (RELP-PCR) assays on circulatingDNA. Gormally et al. detected KRAS mutations in healthy subjectsup to 2 years before cancer diagnosis.43 Comparably, they alsodetected TP53 mutations in the cfDNA of healthy patients on anaverage of 20.8 months before the diagnosis of cancer.43

miRNA. Dysregulated miRNA can affect various cellularpathways leading to tumor development and progression.Hence, miRNA can be used in the diagnosis and managementof cancer.44–46 Circulating miRNA biomarkers provide severaladvantages in liquid biopsy such as high stability, early detec-tion and minimal invasive means for monitoring cancer. Com-pared to other circulating RNAs such as mRNA and lncRNA,miRNAs are more stable and exhibit sturdy expression patternsin clinical samples.8 Different cancers have characteristic sig-natures of the miRNA expression pattern. Recently, a lot ofattention has been focused on the exploration of diagnosticsignificance of miRNA in cancer.47–49 Calin et al.50 demon-strated the underexpression of miRNA 15/16 in chronic leuke-mia by showing a correlation between tissue derived miRNAand cancer miRNA 15/16. Lawrie et al. reported on the diag-nostic significance of circulating miRNAs in B-cell lymphoma.51

In their report, miR-21 and miR-155 levels were relatively highin the serum of cancer patients when compared with healthyindividuals.

Exosomes. Exosomal vesicles play a pivotal role in differentpathological conditions, such as infectious diseases,52 obesity

Fig. 2 Schematic representation of the biogenesis of micro RNA (miRNA): the synthesis of miRNAs and their application in translational repression andtranscriptional modulation. Reprinted with permission.34 Copyright 2012, Elsevier.



and pregnancy complications53–55 and different types of cancer.56,57

Several studies have also highlighted the association of exosomeswith coagulation, angiogenesis, apoptosis and inflammation.58,59

In addition, exosomes shed from tumor cells can transportoncogenetic molecules to recipient cells and regulate their geneexpression, thus playing an important role in the progression,metastasis and drug resistance.35,60–62 In recent years, manyscientists examined the potential of exosomes in the diagnosisand therapeutic interventions of numerous diseases includingcancer, neurodegenerative diseases, infectious diseases andcardiovascular diseases. A number of studies have recentlyindicated the significant diagnostic potential of exosomal proteinsfor breast cancer.63–65 For example, Rupp et al. coupled magneticbeads used for exosome isolation in immune-affinity techniqueswith EpCAM and anti-CD24 and demonstrated the ability ofexosomal CD24 to work as a breast-cancer marker.65 Furthermore,studies have also demonstrated the relative association ofexosomal proteins with other various cancer types includingovarian, pancreas, prostrate and colorectal cancer.66,67 Signifi-cantly increased amounts of exosomal survivin have beenrecorded in prostate cancer patients compared to healthypatients.68 Glypican-1, another surface exosome protein, hasbeen demonstrated to be the only surface protein present inpancreatic cancer serum; nevertheless, the glypican-1 protein isnot found in benign pancreatic disease-derived samples andthese findings strongly indicate the potential of glypican-1 towork as a pancreatic cancer biomarker.69

3.2 CBs as prognostic biomarkers

CTC. Once single CTCs are isolated, a range of genomicprofiling technologies can be applied. In the past few decades,numerous genetic studies have been explored on RNA or DNAderived from enriched CTC cohorts with little sensitivity due totumor profile covering by wild type DNA leukocytes.70,71 SingleCTC analysis prevents the hurdle of leukocyte contamination,thus enabling the analysis of CTC heterogeneity and helps inlocating co-existing mutations within a cell. Several amplificationstrategies for NGS, genotyping and array analyses for singlecells have been established.72–75 Due to the amount of ampli-fication needed for single-cell analysis, many concerns havebeen put forward over the WGA errors dissimulating as ampli-fication bias and somatic mutations or allele dropout distortingthe genomic profiles.76 In spite of the difficulties of single-cellprofiling, such as costly downstream processing, the NGSand WGA strategies have demonstrated to be sturdy enoughto reliably extract similar detailed information from one cellcycle.77

CtDNA. Degeneration and recurrence are a major problemfor monitoring cancer due to minimal residual disease (MRD),which consists of tumor components that remain after chemo-therapy or therapeutic surgery. Currently, there are few availableeffective markers for monitoring MRD in solid tumors despite theroutine monitoring of MRD for hematological malignancies withwell characterized pathognomonic lesions.78 Removal of solidtumors by surgical techniques provides an opportunity to identifyreliable and personalized markers which can be employed in

non-invasive assays for continuous monitoring of recurrenceand relapse.79

miRNA. Over the past few years, a number of studies haveshown the effective prognostic role of miRNA in cancer com-pared to mRNA as a result of its high stability. Schetter et al.demonstrated the significant connection between overexpressedmiR-21 and poor prognostic and therapeutic results in coloncancer patients.80 On the other hand, Takamizawa et al. high-lighted the correlation of downregulated let-7 miRNA with lowsurvival of lung cancer patients.81

3.3 CBs as therapeutic biomarkers

CTC. With the increasing evidence of diagnostic potential ofCTCs in various cancer types, their clinical application stillneeds to be explored. Many studies have demonstrated a strongcorrelation of survival rates and reduced progression freesurvival with CTC baseline levels in many cancer types. These cancertypes include breast, liver, colorectal, gastric and melanoma.1,82

Studies have also highlighted that detection of CTCs in patient’sblood sample after therapy indicates reduced progression freesurvival and overall survival rates.

Exosome. Several exosome-derived therapeutic approaches havebeen developed such as tissue regeneration therapy, drug delivery,vaccine development and gene silencing. The therapeutic potentialof exosomes in tissue regeneration has been demonstrated by Laiet al. using exosomes derived from mesenchymal stem cells.83 Asignificant reduction of infarct size in a mouse model of myocardialischemic injury is shown. More so, exosomes extracted frommesenchymal stem cells (MSC) were recently applied for pediatricrefractory graft-versus-host disease (GvHD) treatment84 and thisfurther triggered their potential utility for other related diseasesincluding type 1 diabetes.85 MSC-derived exosomes in animalmodels were also found to speed up the functional recovery fromstroke and brain injuries. This may be attributed to the improvedtherapeutic neurogenesis and angiogenesis.86,87

4. Isolation and purification of CBs

Many technologies have been developed for isolation and analysisof CBs. However, it is imperative to identify the major obstaclesassociated with their analysis. One of the major obstacles in CBanalysis, particularly CTCs, is their complex surface, and the overallheterogeneity immensely increases their scarcity in circulation.More so, depending on the stage and cancer type, CTCs havevarious sizes, surface protein expressions and physical properties.Also, the identification and characterization of single tumor cellsare considerably difficult to uncover compared to that of othermillions of hematopoietic cells. Thus, highly sensitive and specificanalysis strategies are required to obtain maximum CTC collection.CTC enrichment strategies can be categorized according to thephysical and biological properties of CTCs, as described in Fig. 3.

4.1 Immunomagnetic isolation and enrichment technologies

To date, various technologies have been developed to isolateand detect CTCs. The CellSearch CTCs System is the only



commercial test approved by the Food and Drug Administra-tion (FDA) for the enumeration of CTCs in whole blood. Theimportance of the CellSearch assay results has been extensivelyillustrated through pilot study programs and regulated clinicaltrials. To date, the CellSearch system is regarded as the goldstandard for quantifying CTCs. The system presents a vastlystandardized and automated platform for tumor cell detectionin whole blood. CTC enumeration error is minimized duringsample preparation steps and uses immunomagnetic nano-particles directed against EpCAM to isolate and concentrateepithelial tumor cells.89,90 However, the efficiency of theassay is dependent upon the expression levels of EpCAM andCytokeratin target antigens. These can vary remarkably andalso EpCAM is down-regulated by processes such as EMT.91

Also, non-tumor epithelial cells have been found in the bloodcirculation of patients with prostatitis92 or patients undergoingsurgery.93 Thus, the heterogeneity of CTCs is a major limitationfrom a technical standpoint which resulted in alternativemethods of CTC enrichment, such as the CTC-iChip which isindependent of tumor antigen expression.94

Proteins and receptors present in the exosome membraneprovide an opportunity to develop highly specific strategies toisolate exosomes through the immunoaffinity interactionsbetween proteins and their antibodies, and receptors withspecific ligands. Over the years, numerous immunoaffinity

capture-based methods have been established for exosomeisolation. Recent studies have demonstrated the effective iso-lation of exosomes from antigen cells by utilizing antibodiescoated with magnetic beads. Hence, it is worth noting thatselecting an appropriate exosome membrane marker is one ofthe most critical steps in immunosensing assays. Biomarkersfor exosome immunoisolation are membrane-bound andexposed on the exosome surface. Zarovni et al. developed adetection platform for isolating and quantifying exosomes inserum, urine and plasma based on ELISA.95 The absorbancevalues from ELISA results were used to compare the expressionof known surface markers and to provide quick readouts ofspecificity and yield of exosomes. Compared to ultracentrifuga-tion, greater exosomal RNA amounts were obtained fromthe ELISA. Furthermore, Zarovni et al. developed an immuno-capture based on magnetic particles and the resultantcapture efficiency of antibody-coated magnetic particles wasclosely related to that of ultracentrifugation.95 More recently,a lipid nanoprobe was employed to capture exosomes fromblood plasma and serum-free-cell culture supernatant. Thismethod successfully determined exosomal DNA isolated from19 stage-IV NSCLC patients and allowed the detection of muta-tions in KRAS codons and EGFR exons.96 The advantage ofusing an immunological technique is that it has high specificitydriven by the antibody–antigen affinity interactions. The major

Fig. 3 Schematic representation of enrichment strategies for CTCs based on their biological and physical properties. Immunoaffinity: (A) positiveselection allows immunomagnetic enrichment based on expressed proteins on CTCs; (B) negative selection removes unwanted cells using antibodiessuch as CD45; (C) CTC immobilization; (D) density gradient approaches enabling CTC separation in media based on their densities; (E) filtration based onthe size of the cells; (F) dielectrophoresis (DEP) employs the dielectric charge across the cell membrane of CTCs in a conductive medium; (G) microfluidichydrodynamics using cell inertia to sort CTCs into distinct microfluidic channels. Retrieved with permission from ref. 88. Copyright 2016, Elsevier.



drawbacks of using this approach are that it is costly andrequires various combinations of antigen–antibody and isola-tion procedures.

4.2 Size-based enrichment and isolation technologies

Physical parameters have been successfully used to isolateCTCs through cell separation. Isolation techniques for CTCsbased on their size exploit the different geometrical andmechanical properties between CTCs and blood cells. Thus, itis imperative to have a clear understanding of the physicaldifferences before establishing size-based CTC isolation tech-nologies. An accuCyte-CyteFinder assay isolates CTCs based ondensity. Sensitive identification and analysis of rare cells,and cross-examination of disease biomarkers can be achievedby using this assay.97 A high-throughput Vortex HT chipgenerates laminar fluid microvortices at high flow rates andisolates large CTCs from whole blood samples.98 This methodexpedites the label-free isolation of rare tumor cells. The single-use ScreenCell Cyto device has the ability to isolate rare, fixedtumor cells with a high recovery rate and also permits live cellisolation.99

Size exclusion chromatography has also been used to isolateexosomes. This method separates macromolecules based on theirsize. It applies a column packed with porous polymeric beads.Size-exclusion chromatography allows the precise separation oflarge and small molecules and application of various solutions.Compared to centrifugation methods, the structure of exosomesisolated by chromatography is unaffected by the shearing force.Exosomes can also be isolated by sieving them via a membraneand using filtration by pressure or electrophoresis.

4.3 Differential ultracentrifugation and density gradientcentrifugation

Ultracentrifugation is regarded as the gold-standard exosomeisolation technique.100 This approach is relatively easy to use, isaffordable and does not require technical expertise and samplepre-treatment. With these merits, ultracentrifugation-basedmethods have become a promising alternative for exosomeisolation among researchers. Centrifugation-based methodsinvolve the application of a centrifugal force to an exosomesample solution such as cell culture media or biological fluids.Two types of preparative ultracentrifugation such as differentialcentrifugation and density gradient ultracentrifugation havebeen widely used. Differential centrifugation involves multiplesteps of centrifugation cycles of different centrifugal force andduration to isolate exosomes based on their size differencesand density. The quality and quantity of exosomes isolatedusing differential ultracentrifugation depend on the centrifugalspeed, radius of centrifugation, rotor type, angle of sedimenta-tion, pelleting efficiency, and solution viscosity. Separation ofexosomes by gradient ultracentrifugation is based on their size,mass and density in a pre-built density gradient medium in acentrifuge tube with steadily lower density from bottom to top.Although density gradient ultracentrifugation requires minimalload and extensive centrifugation times, it provides less con-taminated exosomes as compared to ultracentrifugation alone.

The efficiency of differential centrifugation is also subject tooperator-dependent variability.101

4.4 Filtration

Filtration approaches can be combined with ultracentrifuga-tion to improve its relative performance. Ultrafiltration is basedon the separation of resuspended particles using their mole-cular weight and size.102 The filtration step eliminates largedebris and dead cells while the ultracentrifugation step furtherpurifies the filtered samples. Compared to other isolationmethods, the filtration method is easier, faster and does notrequire specialized equipment. However, during filtration,exosomes can be trapped in filter pores and the applied forcethat helps in the passage of the sample through filter mem-branes may damage, deform or break large exosomal vesicles.103

Exosomes entrapped in membranes can be recovered by using amembrane with low exosomal protein-binding properties.A centrifugation step can be used to avoid the force driven stepthereby preventing the deformation of exosomes.

Microfiltration based methods utilizing track-etched poly-mer filters have been used to isolate cells in whole blood.Recently, track-etched polycarbonate filters were used for enrichingand enumerating CTCs from fixed blood samples.104,105 Onanother note, a flexible micro spring array (FMSA) device hasbeen employed for high-throughput enrichment of viable CTCsfrom clinically relevant whole blood samples.106 The perfor-mance of this device is based on the implementation of flexiblepolymer micro springs as effective microfiltration structuresthat enrich CTCs based on their size and deformability whilereducing cell disruption on initial impact. Sample clogging isavoided by maximizing the device’s porous surface area whichincreases the sample capacity.

4.5 Polymer precipitation

Generally, samples of interest are mixed with a polymersolution at lower temperature and optimum salt concentration,followed by incubation at 4 1C overnight. Low-speed centrifuga-tion is therefore used to recover precipitated exosomes. Therecovered exosomes can be resuspended in PBS for further use.The exosome precipitation method is relatively easy to use, canbe scaled to accommodate larger samples, no specializedequipment is required and can be easily applied in clinicalsettings.103 The precipitation method has been widely usedfor isolating several biomolecules, viruses, and other smallparticles. Polyethylene glycol (PEG) is the most commonly usedpolymer in this approach. Studies have shown that commer-cially available kits achieve the highest yield of isolatedexosomes from clinical samples and are simpler and efficientcompared to conventional methods.107,108 Furthermore, thelargest amounts of mRNAs and miRNAs were extracted fromthe subsequent profiling analysis of exosomes isolated byprecipitation.109 During the low-speed centrifugation, exosomespossibly trapped in the polymeric networks generated by theTamm–Horshfall protein are removed by reducing the polymernetworks with dithothreitol. Subsequently, measurement ofexosomes can be performed using CD9 ELISA. Western blot



can be used to examine the purity of exosomal proteins andRNA measured by qRT-PCR.110,111

4.6 Microfluidics-based enrichment technologies

Microfabrication techniques allow the construction of structuresat or below the cellular length scale with eccentric merits for cellseparation. Microfluidic devices enable precise control of fluidflow. The cell capture efficiency is highly dependent on the cell–antibody contacts that can be monitored through the fluid flowrate and direction. Nagrath et al.112 developed the first micro-fluidic device (the CTC-chip) for CTC enrichment from patients’blood with common epithelial tumors. The CTC-chip containedan array of microposts chemically functionalized with anti-EpCAM antibodies. The design and liquid flow were optimizedto enable high cell-capture efficiency by the antibody-coatedmicroposts. 50% purity was obtained when applied to captureCTCs in whole blood samples of metastatic cancer patients withsample concentrations ranging between 5 and 1281 CTCs per mL.

Although the development of microfluidic methods forexosome isolation is at a premature stage, they present promisingopportunities for application in clinical settings. Only smallersample volumes are required with minimal processing times, andthese methods isolate exosomes with high purity. Microfluidictechnologies for exosome isolation are usually used for diagnosticpurposes due to their high sensitivity.113 In addition to theconventional exosome isolation techniques, innovative sortingmechanisms such as electrophoretic, acoustic and electro-magnetic manipulations can be implemented.114 Developedtechnologies for exosome isolation can be classified into threecategories including (i) immunoaffinity, (ii) sieving (e.g. nano-porous membrane), and (iii) trapping exosomes on porousstructures (e.g. nanowire-on-micropillars).

5. Nanomaterials in CB analysis

Nanotechnology has recently become one of the cutting edgetechnologies which generates new sets of materials that offerexceptional attributes for interfacing bio-recognition events withelectronic signal transduction.115 Nanotechnology is described asthe creation and control of matter at a scale of approximately 1 to100 nanometers.116 Numerous applications of nanomaterialsincluding in clinical diagnostics, health monitoring, pharmaceu-tical analysis and environmental monitoring are driven primarilyby their exceptional physiochemical properties. These includehigh surface-to-volume ratio, reactive capacity, biocompatibilityand other desirable functional properties which are not present inbulk materials.117 In biosensor technology, several transductiontechniques have been employed including optical, piezoelectric,acoustic and electrochemical. However, electrochemical methodsoffer distinct advantages. In particular, electrochemical biosen-sors are relatively cheap, highly sensitive, independent of sampleturbidity, and require very simple instrumentation, which makethem suitable for both centralized and decentralized testing.

Nanomaterials can be used in electrochemical biosensors aselectrode surface modifiers and as signaling labels. The modification

of the electrode surface with nanomaterials enables signalamplification via catalytic activity and conductivity, and alsoprovides superficial interactions with chemical and biologicalreagents and targets. Sensitivity, stabilization and other attri-butes on established platforms have been immensely improved.Nanomaterials conjugated with signaling molecules, such asenzymes and redox-active compounds, are often employed aslabels to amplify the generated signal. Different detection modesof nanomaterial-based biosensors can be used such as voltam-metry, amperometry, and impedimetry. The construction offunctional nanoscale electrode materials is recently advancingthe wide application of electrochemical biosensors in liquidbiopsies. Despite the advantages of modifying electrochemicalbiosensor platforms with functional nanomaterials, there areseveral characteristic drawbacks. These include aggregation anddissolution of nanomaterials which impacts the mass transportand electron transfer. However, functional nanocompositescan be grown directly on the electrode surface and this helpsto resolve the recurrence of dissolution and aggregation asproposed by Govindhan et al.117,118

Electrochemical methods represent one of the mostpromising approaches for the detection and determination ofCBs in body fluids, resulting from their rapid responses, lowerdetection limits, diminutive size and ease of manufacture.119,120

Enormous efforts have been devoted towards the expansion ofelectrochemical biosensors based on functional nanostructuredelectrode materials for the detection of CBs in liquid biopsies.Combined with electroanalytical methods, these materialshave practical significance in the development of technologyplatforms for diagnosis, health monitoring and biologicalapplications. Many publications have reported the preparationof biosensors and their application in diagnostic analysisand several biosensors have been constructed using differentmaterials.121,122

Several nanomaterials have been investigated to analyzetheir properties and recent applications in biosensors. Theelectronic and mechanical properties of nanomaterials havebeen explored to enhance biological signaling and transductionmechanisms. Among these are metallic nanoparticles, carbonor metallic nanotubes, magnetic particles, and functionalizedconductive polymers. These can diversely be applied for theimmobilization of bioelements in several biodetection techni-ques. The exceptional properties of metal-based nanoparticlesmake them suitable candidates for electronic and opticalapplications owing to their high catalytic activity, large surfacearea and good biocompatibility. The exploitation of opto-electronic properties of nucleic acid sequences allows efficientdetection using metallic nanoparticles. Enzymatic detection ofglucose using quantum dots as fluorescence agents and con-jugation of colloidal nanoparticles with antibodies enablespecific biomolecular detection in immunosensing and immu-nolabelling applications.

5.1 Magnetic nanoparticles

Magnetic nanoparticles have been widely used in variousapplications such as magnetic separation, biosensing and



thermal ablation therapy. The use of superparamagnetic parti-cles or magnetic beads in biosensing applications has recentlybecome popular. This has been due to advantages such as theirphysiochemical properties, biocompatibility, environmentallyfriendliness and low-cost production. In addition, biologicalsamples show very low magnetic background, and thus highlysensitive measurements can be performed in turbid or other-wise less visible samples without further processing. Magneticnanoparticles exhibit their best performance in the size rangebetween 10 and 20 nm due to supermagnetism.123

Iron oxide superparamagnetic nanoparticles have recentlybeen applied as nanozymes, exhibiting distinctive attributesincluding large surface area, mobility and high mass transference.In addition, they can be retrieved easily by applying an externalmagnetic field. The use of these superparamagnetic nanoparticleswith their excellent environmental biocompatibility represents aneffective green chemistry approach because of their successiverecovery cycles as biocatalysts. Recently, our group synthesizednovel gold-loaded ferric oxide nanocubes (Au-NPFe2O3NC)124 thatexhibit multiple functionalities including superparamagnetism,high electrocatalytic activity and high surface-to-volume ratio foradsorption of targeted nucleotides.125–127 The metal-loaded,highly porous, superparamagnetic nanoparticles have been usedas (i) dispersible capture agents,128 (ii) electrocatalysts,129 and(iii) nanozymes130 (they possess peroxidase-like activity) for thedetection of circulating biomarkers (e.g., cell-free DNA, tumourDNA, microRNA, autoantibodies, exosomes, tumour cells, etc.)in body fluids.

5.2 Polyhydroxyalkanoate (PHA)

Over the past few years, there has been a growing cognizancetowards the potential application of biopolymers in medicine andbiotechnology. In particular, bioengineered nano-microstructuresproduced by microorganisms have gained more interest due totheir functional properties such as biocompatibility, biodegrad-ability and low toxicity which are compatible with biomedical andbiotechnological applications. Biopolyester beads represent acomplex set of polyesters synthesized by most genera of bacteriaand members of the family Halobacteriaceae of Archea131 and aredeposited as water-insoluble cytoplasmic nano-sized inclusions.PHA beads are spherical with a polyester core surroundedby proteins. A key enzyme, polyester synthase, catalyzes theenantio-selective polymerization of (R)-3-hydroxyacyl-coenzymeA thioesters to polyesters. Ralstonia eutropha polyester synthaseprotein PhaC is the most characterized PHA synthase for thisprocess. Polyester chains are self-assembled, thus forming poly-mer granules that have a hydrophobic core. The PHA synthaseprotein remains covalently attached to the synthesized polyestersurface.132 The resultant spherical granules have different sizesranging from 50 to 300 nm and occupy the intracellular space.133

The structure of PHA granules has not been fully established.134

The amorphous mobile state of the hydrophobic polyester core,in vivo, consists of water which acts as a plasticizer to preventcrystallization.135 This mobile state allows the synthesis anddegradation by enzymes. The biosynthesis mechanism of PHAvia the polymerization of (R)-3-hydroxyacyl-CoA enables the

generation of spherical inclusions. These begin to assembleas the PHA synthase facilitates the formation of an insolublepolymer with higher molecular weight from soluble substratemonomers. The synthase is covalently attached to the polyesterchain and during polymerization, it continuously assimilatesthe monomer substrates into the nascent polyester chain untilthe polymerization is terminated as a result of the depletionof the substrate or lack of space in the cell. The resultantspherical granules’ size and the number of inclusions per cellvary among organisms with the diameter in the range between100 and 500 nm and between 5 and 10 granules per cell.136

Several proteins play a crucial role in the synthesis, degrada-tion and formation of PHA granules137 and these have beenclassified into four main categories. These are, PHA synthases(PhaC), depolymerases (PhaZ), regulatory proteins (PhaR),and phasins. The PhaC enzyme plays a crucial role in PHA bio-synthesis through its catalytic activity towards (R)-3-hydroxyacylCoA thioester substrates. PHA depolymerase enzymes facilitatethe degradation of amorphous PHA within granules and aid inassembling PHA granules as a source of energy.138 Phasins arenoncatalytic proteins mostly found on the surface of PHA whichstabilizes the PHA granules and prevents the merging ofseparated granules.139 Phasins also regulate the unspecificbinding of unrelated proteins on the granule surface whichcan inhibit the overall cell metabolism.139 The transcriptionalPhaR monitors the production of phasins and the generation ofPHA granules.

To date, several biomolecules have been successfully immo-bilized on the surface of PHA granules. This shows thepotential biomedical application of these bacterial storagecompounds for diagnostic and therapeutic uses. Recently,functionalized nano-/micro-beads have been produced byexploiting the covalent binding of the PHA synthase proteinto the surface complemented by the high stability of PHA beadsoutside the cell. Functional proteins of interest are generated asgenetic fusions to the PhaC synthase protein. During PhaCprotein expression in cells, granules form with the fusionproteins displayed stably on the surface of the granule, thusenabling the production of functionalized beads in a singlestep. This process produces engineered biopolyester beadswhich are capable of binding to immunoglobulin (lgG),140,141

inorganic substrates and biotin,142,143 displaying target antigens,144

and enabling the production of purified proteins145 and diagnosticimaging.146,147 The exceptional properties of the granules suchas biodegradability and biocompatibility make them attractivereplacement for beads generated by chemical means (Fig. 4).

6. Detection technologies of CBs6.1 Amplification and sequencing-based approaches

PCR based methods have been the most commonly usedtechniques for CTC detection since their emergence in the1990s. Primers that selectively amplify the translocationlesion are selected in the flanking regions of the translocation.RT-PCR and qRT-PCR methods are regarded as the most



sensitive for detecting transcripts characteristic of tumor cells.The detection sensitivity of these techniques is valued at onemalignant cell out of 1–10 million other cells: one cancer cell in1–10 mL of whole blood.148 In addition, high specificity isattained by designing primers that are specific for the gene ofinterest, and high efficiency is generally achieved as a result ofthe simultaneous analysis of the entire genomic DNA or RNA inone reaction.

RT-PCR is a rapid and inexpensive method for amplificationof nucleic acids. However, its sensitivity is very low for detectingmutations in the background of wild-type DNA, with 10–20%allele frequency.149 Various PCR-based techniques have beendeveloped to improve the sensitivity including Allele-Specific

amplification (AS-PCR),150 Peptide Nucleic Acid-Locked NucleicAcid (PNA-LNA) PCR,151 and co-amplification at lower dena-turation temperature (COLD-PCR).152 The driving force forthese assays is mainly centered on blocking the amplificationof the normal allele using a blocking oligo at the 30 end. Theblocking allows the amplification of the mutant allele tooccur. During the PCR, the modification step that allows theenrichment of variant alleles from a mixture of mutationcontaining DNA and wild-type DNA is also used by thesemethods to amplify the target nucleic acids.

Digital PCR (dPCR) is a sensitive and robust platform that isused to detect point mutations in ctDNA at low allele fractions.The commonly used dPCR platforms include droplet-based

Fig. 4 Various applications of PHA granules. (A) Different synthesis approaches for functionalized PHA beads. (1) Recombinant production of a plasmidencoded fusion of the target protein and a GAP in a PHA synthesizing host strain. (2 and 3) Natural PHA producing organisms generate native PHAgranules which are subsequently isolated by chemical extraction followed by in vitro bead generation. Finally, the purified GAP fusion protein binds to thePHA granules/beads in vitro (2 and 3). (B) Schematic review of various biomolecules and compounds that have been successfully functionalized on thesurface of PHA granules. Adapted with permission from ref. 136. Copyright 2009. American Chemical Society.



systems, microfluidic platforms for parallel PCR such as dro-plet digital PCR (ddPCR), and BEAMing (beads, emulsions,amplification, and magnetics).153 Droplet dPCR applies thesame principle of dPCR, whereby single DNA molecules aredispersed into thousands of droplets. Droplets containingmutated or wild-type DNA strands can be captured and enum-erated via a flow cytometry apparatus by using fluorescentlylabelled TaqMan probes.154 DdPCR sensitivity relies on thequantity of droplets.155 The commercially available ddPCRsystem, QX200 Droplet dPCR System, is capable of producing20 000 nanolitre-sized droplets. This number has been ampli-fied by using the RainDrop dPCR System, which has the abilityto produce up to 10 million picolitre-sized droplets. However,the ddPCR method has limitations including long dropletprocessing times and difficulties in producing droplets.156,157

The BEAMing approach incorporates multiple techniques toimprove the specificity and sensitivity of mutation detection.Compared to emulsion and flow cytometry, rare mutations canbe detected effectively with AF less than 0.01%.158 BEAMing iseasy to set up and can be executed using readily availableapparatus such as flow cytometer, magnetic stirrer and thermo-cycler. The sensitivity of the BEAMing approach can beincreased by examining more recyclable beads which containvariant alleles.159 Several sensitive targeted deep sequencingmethods have been developed for the analysis of ctDNA specificgenomic areas. These include Ion-AmpliSeq,160 safe-sequencingsystem (Safe-Seq),161 tagged amplicon sequencing (Tam-Seq)162

and cancer personalized profiling by deep sequencing (CAPP-Seq).75 Amplicon sequencing is mainly used for evaluating mutatedgenes in specified genomic regions. The Ion-AmpliSeq method israpid, cost effective and requires small amounts of input DNA.

However, it suffers from a high error rate in detecting smallinsertions and deletions. Targeted capture-based platforms arewidely used for examining gene changes in cancer.75

In the case of miRNA analysis, RT-qPCR offers several meritssuch as sensitivity, a wide dynamic range, less sample inputand better accuracy.163 This method is based on the reversetranscription of RNA to cDNA followed by a quantitative poly-merase chain reaction and the accumulation of the reactionproduct is followed in real time. Since the discovery of miRNA,multiple RT-qPCR-based miRNA expression analysis strategieshave been developed. The TaqMan technology,164 the commonlyused method for miRNA analysis, utilizes a stem-loop reversetranscription primer system to reverse transcribe the RNA andamplify cDNA (Fig. 5). This approach is generally employedto recognize global differences between miRNA expressionsacross comparative samples. The TaqMan approach enablesthe quantification of the targeted miRNA expression to validatethe results obtained by whole-genome screening to determine afew specific miRNAs in a large sample cohort. Le Carre et al.described the validation of a multiplex reverse transcriptionand pre-amplification of miRNA using TaqMan method onhuman muscle plasma samples. The method could screen alarger number of miRNAs in parallel.165 Despite the highsensitivity and efficient quantification of relative miRNA con-centrations using the RT-qPCR, absolute quantification ofRNAs is still limited and only smaller number of expressedRNAs are required. This is not suitable for high-throughputmiRNA screening.163,166

Sequencing technologies comprise several steps widelyclassified as template preparation, sequencing and imaging, anddata analysis. The coalition of specific procedures differentiates

Fig. 5 Methods of miRNA detection and quantification by RT-qPCR. (a) Reverse transcription of a single miRNA using a gene-specific stem-loop primer.A mixture of specific primers and a hydrolysable probe are then used to amplify the resultant cDNA. The Taq polymerase displaces and hydrolyses theprobe to separate the fluorophore and quencher. (b) miRNAs are polyadenylated by poly(A) polymerase followed by reverse transcription using an oligodT priming strategy. The resulting cDNA is amplified using specific primers and the reaction is monitored in real-time using a fluorescent dye.Reproduced with permission.166 Copyright 2014, Biological Procedures Online.



each technology and determines the output data of eachplatform. The next-generation sequencing allows the measure-ment of miRNA in a genome-wide fashion.167 NGS is capableof identifying and quantifying both known and unknownsequences simultaneously and also allows multiplexed expres-sion analysis of miRNA from various sample sources in a singleexperiment. This prevents factors that may have a negativeimpact on test results. Sequencing-by-synthesis technology isone of the NGS technologies that is widely used for thedetermination of novel miRNA signatures or panels due tothe broad depth sequence data it provides which allows exactquantification of miRNA species. In this method, purifiedmiRNA is ligated to adaptors at both 30 and 50 ends followed byconversion into complementary DNA (cDNA). Index sequencesare then introduced into cDNA during the subsequent amplifica-tion step that tag the cDNA library. This later allows parallelquantification of several samples in a single experiment.Amplification has generally been conducted using either emul-sion PCR (emPCR) or bridge PCR (bPCR). Libraries of cDNA arethen introduced to a flow cell where capturing and subsequentbridge-amplification of single cDNA occurs, thus resulting inclonal spots of each cDNA generated in the flow cell. The Sangermethod is then applied to the flow cell to sequence the respectivespots using fluorescently labelled dye terminators coupled with aCCD camera. NGS approaches have a broad range of applica-tions. Recently, they have been used to characterize the evolu-tionary relationships of ancient genomes168,169 and to unravelthe role of non-coding RNAs in health and disease. AlthoughNGS has been used for discovery and validation of miRNAsignatures, it could also be employed for measuring smallmiRNA panels from a larger cohort of samples similarly toroutine testing in a clinical laboratory. However, when comparedto qRT-PCR, NGS is still an expensive and time-consumingmethodology since it is not fully automated. More so, the dataanalysis of NGS is not yet standardized and its routine clinicalapplication is still questionable.

6.2 Microfluidics based technologies

Microfluidics is a technology that is characterized by manip-ulation of fluids at submillimeter scale and has demonstratedgreat potential for improvements in diagnosis and molecularbiology research. Microfluidics has recently become an attractivealternative to conventional experimental approaches due to itsrapid sample processing and precise monitoring of fluid flow.Several microfluidic platforms have been developed for thedetection of CTCs, ctDNA, miRNA and exosomes. Microfluidicplatforms for CTCs use confocal fluorescence microscopy drivenby a central self-organized array of superparamagnetic particleswithin the microchannel which allow the generation of a sturdyarray. Another microfluidic device, CTC chip, is composed of anarray of microspots containing EpCAM antibodies and canisolate CTCs from blood with high efficiency and reproducibility.EpCAM is overexpressed in a myriad of carcinomas includingthose of the lung, breast, liver, colorectal, prostate and breast,and is not present in hematological blood cells and this allowshighly sensitive capture of CTCs in blood using the chip. The

blood sample is introduced across the chip through a controlledflow system, enabling EpCAM-positive cells to bind to the micro-posts which are subsequently quantified using a camera. Onemajor technical limitation of the CTC-Chip is the reliance onlaminar fluid flow which only allows limited cell–substrateinteractions. To overcome this challenge, a high throughputmicrofluidic mixing device, Herringbone-Chip, with an enhancedCTC isolation platform has been utilized.170 The Herringbone-chip design enables passive mixing of blood cells by generatingmicrovortices which increases interaction between cells and anti-body coated channel walls.

DNA microarrays have been broadly used for detecting targetnucleic acids via sequence-specific hybridization with knownprobes immobilized on solid substrates. High aspect ratio nano-pillar arrays on a silicon-based chip surface have been used toincrease signal intensity in DNA microarrays.171 The structuraldesign of the nanopillar array enables increased probe immobiliza-tion capacity due to their high surface-density platform resulting inincreased target accessibility with minimal background noise.

Inertial microfluidics has also been applied in CB analysisowing to its unique advantages in particle manipulation.172

Particles of interest flow in straight and curved channels andthe inertial lift force is generated leading to the lateral migra-tion to the dynamic equilibrium position. Particle focusing andseparation is achieved as a result of the inertial lift force, bywhich optimum throughput manipulation can be attained. Thecurved channel adjusts the equilibrium position of particles,and particles with different sizes can clearly be separated.

Over the past decade, numerous microfluidic technologies forisolating and detecting exosomes have been developed with severalmerits including high yield, rapid analysis, high efficiency, lowsample input and automation.173,174 With these merits, microflui-dic platforms have demonstrated great potential for exosomeanalysis in clinical settings. Zhao et al. recently developed a simplemultiplexed microfluidic approach using immunomagneticbeads.175 When employed in the blood-based diagnosis of ovariancancer by multiplexing measurement of three exosomal tumormarkers (CA-125, EpCAm, CD24), the ExoSearch chip showedsubstantial diagnostic power and was comparable with the stan-dard Bradford assay. Vaidyanathan et al.176 also reported a simplemultiplexed microfluidic assay for detecting various exosomesbased on a tunable alternating current electrohydrodynamicmethod. The approach exhibited high sensitivity for analyzingexosomes derived from cells expressing HER2 and PSA comparedto hydrodynamic based assays (Fig. 6). Despite the recenttechnological advancement in microfluidics, there are still manychallenges hindering the clinical application of microfluidics-based technologies. Several microfluidic chips without automa-tion need manual off-chip sample preparations which thereforelimit their real application in clinical settings. Furthermore,experienced personnel are required to conduct the experiments.

6.3 Immunoassay methods

6.3.1 ELISA. The ELISA detection-based principle usuallyinvolves the direct immobilization of circulating exosomes to amicrowell plate followed by blocking with a blocking agent.



After blocking, a recognition antibody is introduced to bind tospecific antigens present on the CB surface. Lastly, an HRP-tagged antibody is used for specific and sensitive readout via anenzymatic signal amplification step. A colorimetric substratesuch as TMB is used for the assay readout.177,178 Logozzi et al.designed a sensitive and specific sandwich ELISA to measureexosomes in plasma and cell culture media using housekeepingproteins CD63 and Rab-5b and caveolin-1 (tumor-associatedmarker).60 One major limitation of the ELISA-based detectionapproach is the high nonspecific adsorption of biomoleculesfrom complex body fluids during exosome detection.

The ELISA mechanism has been widely adopted to analyze amyriad of CBs. Recently, Boriachek et al.179 reported on directexosome isolation and subsequent detection with calorimetricand electrochemical-based methods utilizing the ELISA detec-tion principle. In this approach, gold-loaded nanoporous ferricoxide (Au-NPFe2O3NC) nanoparticles were functionalized withexosome associated antibodies (CD9 or CD63). Upon magneticisolation and purification, exosome-bound nanoparticles wereimmobilized onto a placenta alkaline phosphatase (PLAP)antibody-modified screen-printed electrode. The oxidation ofTMB in the presence of H2O2 was catalyzed by Au-NPFe2O3NCand the observed color change indicated the presence of PLAP-specific exosomes in the Au-NPFe2O3NC/CD9/exosomes/PLAPimmunocomplexes.

6.3.2 CellSearch. The CellSearch system is the only clinicallyvalidated and FDA cleared test for capturing and enumeratingCTCs in blood samples. It uses magnetic beads coated with anti-EpCAM antibodies for capturing and enumerating epithelialCTCs. The captured tumor cells are then treated with a nuclear

stain (DAPI) and identified by cytokeratin staining fluorescentantibody conjugates against epithelial markers EpCAM andcytokeratins. Non-specific staining of hematopoietic cells isexamined by counterstaining with leukocyte marker (CD45)antibodies. Automated fluorescence imaging capable ofclassifying epithelial cells positive for cytokeratin and negativefor CD45 is then used to analyze the captured cells. Severalreports have indicated the clinical significance of the CellSearchsystem in the analysis of colon, breast and prostate cancer.180–183

6.3.3 EPISPOT. This method is based on the ELISA andspecifically detects CTCs and disseminated tumor cells (DTCs)in cancer patients. The technique is based on the detection ofproteins produced by functional CTCs and DTCs combinedwith a negative enrichment. EPISPOT avoids direct contact withtarget cells and detection of CTCs is based on dischargedproteins during short-term culture. Only viable cells are detectedafter the depletion of CD45 positive cells.184

6.3.4 Immunocytochemical methods. Over the past fewyears, the immunohistochemistry approach has been widelyused to analyze CTCs and DTCs using related specific andsensitive antibodies to isolate and detect. However, the choiceof CTC markers is still a major concern with the majority ofcancers lacking specific tumor or organ markers. Thus, the lackof reproducibility, reliability and specificity of immunochemistrypaved the way for alternative pathways for CTC detection basedon immunocytological methods.

6.4 Electrochemical based approaches

In the electrochemical detection approach for exosomes, bio-recognition molecules bind to the target exosomes and

Fig. 6 Schematic representation of multiplexed device functionalization, capture and colorimetric detection of captured exosomes driven by tunablealternating current electrohydrodynamic-induced nanoshearing. Adapted with permission.176 Copyright 2017, American Chemical Society.



selectively recognize antigens present on the exosome surface.Detection antibodies are labelled with electroactive moleculesand used as an electroactive transducer.185 Several electro-chemical assays have been developed for exosome detectionin previous years. A recently developed exosome platform(iMEX) is a miniaturized analytical system that can rapidlyisolate and detect exosomes in clinical specimens186 (Fig. 7).The iMEX system allows parallel quantitative measurementsand can directly isolate cell-specific exosomes from plasmasamples with high sensitivity. The iMEX system has two mainsteps which are magnetic selection and electrochemicalsensing. Firstly, immunomagnetic beads are coated with anti-bodies against CD63 and used to capture and isolate exosomes.Secondary antibodies tagged with HRP are then addedwhich facilitate the catalytic oxidation of TMB to generate anelectrical current. The entire assay is conducted within an hourwith a minimum amount of the input sample consumed.Substantial progress has been made over the years towardsthe integration of nanotechnology with electrochemical bio-sensing strategies for the analysis of exosomes. Our group hasrecently demonstrated a new method for quantifying exosomesbased on quantum dot (QD)-functionalized disease-specificantibodies.187 The assay involves three steps where initially thebulk exosomes are magnetically captured by magnetic beadsfunctionalized with a tetraspanin CD63 antibody followed bythe quantification of breast and colon cancer-related exosomesusing CdSe QD-functionalized biotinylated breast and colon

cancer-related antibodies. This method exhibited an LOD of100 exosomes per mL.

Electrochemical detection methods for miRNA usually relyon hybridization between the surface bound reciprocal receptorprobe and target miRNA on the electrode surface. The hybridi-zation between miRNA and reciprocal probes generates aquantifiable signal. Detection of miRNAs is mostly performedvia voltammetric, amperometric and impedimetric methods.15

Owing to the smaller size of miRNAs, the detection sensitivityof electrochemical platforms can be enhanced by labelling thetarget miRNA and coupling with electrocatalytic amplification.Transduction is driven by the electrochemically active reporterspecies whose properties can cause changes in interfacialproperties as a result of miRNA hybridization.

The commonly used reporters are enzyme-substrates andsmall molecules. Gao and Yang reported on the application ofelectrocatalytic nanoparticle tags for the detection of miRNA.188

Their approach utilizes an indium oxide electrode as animmobilization platform for oligonucleotide capture probes.After hybridization, isoniazid-capped OsO2 nanoparticles areintroduced to the electrode via a condensation reaction tochemically amplify the signal. The nanoparticles effectivelycatalyze the oxidation of hydrazine. The successful and wideapplication of enzymes as labels is centered on their ability toconvert single hybridization events into multiple moleculesthat could be detected. Recently, numerous papers reportedon the application of enzymatic reaction for recognition of the

Fig. 7 Schematic representation of an iMEX platform. (A) Schematic of the sensor. The sensor can quantify signals from eight electrodes simultaneously.Exosomes are captured immunomagnetically by small cylindrical magnets located below the electrodes. (B) Circuit diagram with eight potentiostats witheach consisting of three electrodes: reference (R), counter (C), and working (W). (C) A packaged device. (D) iMEX assay representation. Exosomes labelledwith HRP are captured on the magnetic bead surface coated with antibodies against CD63. Eight channels are examined simultaneously using TMB.Reprinted with permission.186 Copyright 2016, American Chemical Society.



hybridization event between the targeted miRNA and probe.Yanli et al.189 reported on the development of an ultra-sensitivetetrahedron-based electrochemical miRNA sensor based on enzymetransduction. In this approach, tetrahedral nanostructure-basedcapture probes complementary to part of the target miRNA wereself-assembled on the gold surface and detection was performed ina sandwich assay format. The signal probe 2 flanked with a biotintag binds specifically to poly-HRP80 and in the presence of 3,30,5,50-tetramethylbenzidine (TMB) catalyzes the reduction of H2O2. Thismethod exhibited excellent real applicability in the analysis of realsamples (o1000 copies of miRNA).

6.5 Optical methods

6.5.1 Surface plasmon resonance (SPR). Nanoplasmonic sen-sing approaches are optical methods that rely on local refractiveindex changes from a minute sensing volume producing a signalreadout, often in the form of an optical spectral shift.190 Nano-plasmonic methods are commonly label-free and can be easilydesigned to enable multiplexing detection using small input

sample volumes. SPR has recently emerged as a versatile tech-nique for analyzing molecular interactions by evaluating therefractive index change on a chip surface. The SPR biosensingtechnique involves the functionalization of the surface of a metalfilm supporting a surface plasmon with specific biorecognitionelements, for example, complementary nucleic acid strands orantibodies. Molecules of the analyte that are in contact with theSPR sensor bind to the biorecognition element, increasingthe refractive index at the sensor surface which is quantifiedoptically. The change in the refractive index exhibited by thecaptured biomolecules is highly dependent on the target concen-tration and its properties. During the measurement, the angularshift in the SPR spectrum is monitored over time and reveals theamount of the bound target. In previous years, several nano-plasmonic techniques have been developed for detecting CBs.Xue et al. developed an SPR sensor based on two-dimensionalantimonene nanomaterials for detecting cancer-related bio-markers, miRNA-21 and miRNA-155. This approach exhibited ahigher sensitivity with an LOD of 10 aM (Fig. 8). SPR offers

Fig. 8 (i) Fabrication of antimonene materials. (ii) A schematic representation of miRNA sensor integrated with antimonene nanomaterials. (I) Assemblyof antimonene nanosheets on gold film surface. (II) Absorption of gold nanorods modified with single stranded DNA (AuNR-ssDNA) on antimonenesheets. (III) Formation of double strands with complementary AuNR-ssDNA by passaging different concentrations of miRNA solution. (IV) Release ofAuNR-ssDNA on antimonene nanosheets as a result of the interaction with miRNA. The AuNR-ssDNA molecular reduction on SPR surface decreasessubstantially the SPR angle. Reprinted with permission from ref. 192. Copyright 2019, Nature Communications.



several advantages such as low cost, high reproducibility, andnon-destructive evaluation of the sample which allows detectionfor both clear and colored samples.191,192 Carrascosa et al.193

developed a label-free biosensing technique for regional DNAmethylation detection (MCF7 cancer cells) using surface plas-mon resonance coupled with molecular inversion probes (MIPs).The SPR signal was a function of concentration with a minimumdetectable methylated target concentration of 100 nM for syn-thetic targets. The coupling of MIPs and SPR has the potential toenhance the sensitive and specific measurement of regional DNAmethylation in a real-time and label-free manner.

6.5.2 Surface-enhanced Raman scattering (SERS). Opticalsensors based on SERS have recently emerged as robusttechniques for detecting biomolecular interactions.194 SERS isa non-invasive method that is commonly used to investigate theconformational changes in molecules under several extrinsicconditions. SERS integrates the structural specificity andexperimental flexibility of conventional Raman spectroscopyto provide an ultrasensitive detection platform for bio-molecules with multiplexing capabilities.195 SERS requires asmall input sample with minimal sample preparation. Alter-natively, SERS labels can be coupled to target-specific ligandsand be used for the selective detection and localization of thecorresponding target molecules. Well-defined nanoplasmonicstructures with exceptional physiochemical properties are cri-tical for SERS. Generally, three platforms are commonly used asSERS-active substrates for biomedical applications: 2D nano-structures, colloidal nanoparticles in solution and colloidalSERS tags196 (Fig. 9).

Colloidal substrates and 2D nanostructures are usuallydesigned to detect the inherent analyte SERS signals, whereasthe nanoparticle tags are coupled with antibodies or other targetligands for detection. Wang et al.197 developed a multiplexedhomogeneous platform to detect miRNA-21 and miRNA-34abreast cancer biomarkers based on inverse molecular sentinelnanoprobes. The modified nanoprobe approach was basedon the nonenzymatic strand-displacement process and the con-formational change of stem loop (hairpin) oligonucleotideprobes upon target binding. A robust and sensitive SERS-basedassay for duplex detection of pathogen antigens with nanoyeastsingle-chain variable fragments has been demonstrated byWang et al.198 Silica coated-purified SERS nanoparticle clusterswere used which enabled sensitive duplex antigen detection.Major challenges associated with SERS-based immunoassaysinclude nonspecific adsorption and the time consumingprocedure. To address these challenges, the same group hasreported on a rapid and simple approach using alternativecurrent electrodynamic-induced nanoscaled surface shearforces to enhance the capture and fluorophore-integratedgold/silver nanoshells as SERS tags. The nanoscaled physicalforces acting within the nanometer range from the electrodesurface increased the sensitivity with a limit of detection of10 fg mL�1 for specific detection of EGFR2 in breast cancersamples (Fig. 10).199

6.5.3 Flow cytometry. Flow cytometry is one of the mostcommonly used methods for quantification and high through-put analysis of exosomes. This method is based on the passageof individual exosomes through a laser spot and measurement

Fig. 9 (a) A simplified illustration of surface enhanced Raman scattering (SERS) arising from the interaction of a laser beam with molecules directlyadsorbed on the surfaces of nanoparticle clusters. (b) Structural design of biofunctionalized SERS nanoparticle tags, composed of a metal nanoparticlecore, adsorbed Raman reporters on the metal surface (green stars), a biocompatible layer (orange layer), and targeting ligands. Adapted with permissionfrom ref. 196. Copyright 2015, American Chemical Society.



of the emitted scattered and fluorescent light. Initially, a sheathof fluid is hydrodynamically focused on the suspended singleparticle to intersect with the laser200 and the resultant signalsare obtained from the corresponding detectors. Flow cytometryenables individual exosomes to be resolved and the measure-ment of several surface markers per exosome.201 The signals arethen amplified and converted to a digital form. However, due tothe smaller size of exosomes, it is difficult to recover weak signalsand only particles larger than 300 nm can be resolved.202

Fluorescence-activated cell sorting (FACS) is a specialized typeof flow cytometry that allows the sorting of exosomal vesiclesbased on fluorescent labelling.203 This approach utilizes specificantibodies tagged with fluorescent dyes to capture and sort targetexosomes depending on the required parameters. Recently, Rimet al.204 developed the FACS method for quantitative measure-ment of exosomes in M1g2908 murine lung fibroblasts and LA-4and KLN 205 murine lung cancer cells. In this approach,exosomes were isolated using CD9- or CD63-antibody-coatedmagnetic beads and analyzed using FACS. An increase inCD63-specific exosomes in LA-4 lung cancer cells was observed.

6.6 Conventional methods

6.6.1 Nanoparticle tracking analysis (NTA). NTA is themost widely used method for characterizing the size andconcentration of exosomes. The size distribution of biologicalparticles ranging between 50 nm and 1 mM can be measuredusing NTA. Exosomal particles are first irradiated by a laserbeam and the resultant scattered light is monitored using anoptical microscope and then analyzed by image processingsoftware. The software monitors the diffusion rate of exosomesthrough the field-of-view. The diameter of the exosome isderived from its rate of Brownian motion which can be relatedto particle size using the Stokes–Einstein relationship which isdependent on the temperature and viscosity of the suspended

fluid.205 Although NTA is capable of analyzing large quantitiesof vesicles compared to electron microscopy and atomic forcemicroscopy, it is still limited to clinical applications.206 Theselimitations are as a result of the lengthy procedures involved indata collection and analysis. Long analysis time may result inbleaching of the fluorescent dye and this tool cannot be appliedfor the analysis of biochemical composition of exosomes.

6.6.2 Northern blotting. Northern blotting (NB) has beenapplied for lin-4 miRNA detection which is a negative regulatorof lin-14 protein.207 The technique combines an electrophoreticseparation – usually by denaturing urea polyacrylamide gel –followed by a semidry capillary transfer to a positively chargednylon membrane. MiRNAs are then hybridized with labelledprobes and imaged. The size separation step in NB allows theapplication of the technique in quantitative expression analysisof primary or precursor miRNA and mature RNA as well as sizevariation of isomiRNAs from imprecision of Drosha and Dicercleavage in upstream biogenesis of mature miRNA.208,209 However,NB suffers from several limitations including low throughput, therequirement of high input RNA and lower sensitivity.210,211

The commonly used labelling system in NB, i.e., radioisotopes(32P), presents several health and safety concerns for researchersand the environment. Also, the use of radioisotopes is timeconsuming and in some instances radioisotope labels need tobe exposed for days in order to detect weak signals. To circumventthe safety concerns of using radioisotopes as labels, hapten-labelled probes coupled with enzymatic detection techniques havebeen established.212 Locked nucleic acids (LNAs) have also beenused as NB probes to enhance the sensitivity and improvemismatch specificity.213 RNA can also be cross-linked to themembrane using 1-ethyl-3-(3-dimethylaminopropyl carbodiimide)(EDC) and an approximately 20-fold increase in sensitivity hasbeen reported by Pall et al.211 Modifications of the traditional NBprocedure allows its application in any laboratory setting.

Fig. 10 Schematic illustration of alternative current electrohydrodynamic surface-enhanced Raman scattering (ac-EHD SERS) immunoassay. Repro-duced with permission from ref. 199. Copyright 2015, American Chemical Society.



6.6.3 Microarray. Detection of miRNA using microarrays isbased on the sensitive and specific hybridization of the targetmiRNA to complementary DNA probes. Microarrays rely on thespatial arrangement of complementary capture probes on asolid substrate, which hybridize with the target of interest.Several microarray platforms have been used to quantify genome-wide RNA including GeneChip (Affymetrix), miRCURY LNA (Exiqon),and SurePrint (Agilent). Microarray-based methods can measuremultiple miRNAs simultaneously, but their clinical utility is limitedby specific probes required, specialized equipment and hybridiza-tion procedures which limits a specific target to confined spots thatare easily observed with fluorescence or imaging instrumentation.The whole chip is subjected to the same hybridization conditionssince all target sequences are analyzed in parallel on a microarray.More so, the hybridization procedure increases the cost andminimizes the reproducibility between platforms.214,215 Analysisof microarray data is time consuming and data normalization isoften difficult (Table 1).

7. Grand challenges associated withCB analysis7.1 Biological challenges

During cancer progression, many tumors discharge biomarkersinto blood circulation, and their analysis offers the potential topredict, screen and monitor therapeutic responses and tumorresponses. The precise quantification of CBs has been greatlyinfluenced by many biological limitations including theirrelatively low abundance in complex biological matrices andnonspecific adsorption of biomolecules.

7.1.1 Low abundance. The study of CBs in clinical speci-mens presents the basis for establishing non-invasive liquidbiopsies. Evidence from clinical studies suggests that indivi-duals with metastatic lesions are more susceptible to haveCTCs that can be easily isolated. However, they are usually verylow in abundance, ranging between 1 and 10 CTCs per mL ofwhole blood,24 which also contains about 7 � 106 and 5 � 109

white blood cells and red blood cells respectively.25 Therefore,technologies that can effectively isolate a single CTC from thebackground of several blood matrices are essential. A consider-able number of novel technologies have been developed such aspositive and negative selection, or free-enrichment assays toeffectively isolate, quantify and characterize rare CTCs.

CTCs can be enriched positively or negatively based on theirbiological properties. The positive enrichment methods selectcells with CTC-like properties that are not displayed by otherblood cell components. This is done through the use of an anti-epithelial antibody, an anti-mesenchymal antibody or an anti-epithelial and anti-mesenchymal antibody. The epithelial celladhesion molecule (EpCAM) is the commonly used surface cellmarker for positive enrichment of epithelial CTCs via immuno-magnetic bead separation, where the antibody-labelled ferro-particles capture CTCs in a magnetic field. Negative enrichmentis achieved by exploiting antibodies against CD45 to removethe redundant leukocytes. Physical characteristics such as size,

deformability, and differences in densities and surface chargescan also be used to enrich CTCs through membrane andfiltration-based systems. However, patients with benign colondisease have been reported to have circulating epithelialcells,270 and these cells might result in false-positive findings.In addition, the possible transition of epithelial-to-mesenchymalcarcinoma cells results in decreased expression of epithelialmarkers that might be the root of false-positive findings. Hence,the identification of additional mesenchymal markers upregulatedduring EMT on CTCs is a prerequisite.

The paltry concentration of cfDNA in the plasma of healthyindividuals, usually between 0 and 100 ng mL�1,153 makes thedetection of ctDNA in plasma samples immensely difficult,requiring more blood samples. Although the concentration ofcfDNA in cancer patients may be 50 times higher than thenormal levels, the amount of ctDNA in the bulk cfDNA isproportionately low. With the influence of tumor heterogeneityresulting from clonal evolution of cells containing tumorinitiating molecules, the selection of branch mutation for ctDNAdetection may not provide accurate results representing the overallctDNA level. However, sensitive detection and quantification ofctDNA has been improved due to the recent advancements in digitalgenomic strategies and parallel sequencing technologies.271,272

Direct analysis of ctDNA through hybridization is often difficultdue to a number of processing steps. These steps include theefficient denaturation of dsDNA into ssDNA which is suitablefor hybridization analysis, and also renaturation of ssDNAmolecules affects the efficiency of the hybridization-basedassay.273 To circumvent this challenge, many strategies havebeen posited including the use of DNA clutch probes (DCPs)that inhibit the renaturation of denatured DNA molecules thusenabling effective hybridization analysis.274

7.1.2 Nonspecific response from biomolecules. Sampleheterogeneity can affect the efficient analysis of CBs. For analysisof exosomes, many factors, including age, gender, and bodymass index, are known or suspected to influence vesiculation,which in turn affects the selection of controls for heterogeneoussamples.275 Therefore, more systematic interrogations arerequired to examine the upshot of sample heterogeneity on theamount, functionality and biogenesis of exosomes.

Circulating miRNAs are often found in complex biologicalmatrices that are generated from various nonspecific cells andother biomolecules. Depending on the detection strategy, thematrix to be used may contain sizeable amounts of proteins,nucleic acids and other biomolecules that could interfere byadsorbing on the sensing surface non-specifically and givingsubstantial background readings. Thus, there is a need forthe development of detection strategies that have antifoulingsensing surfaces to circumvent the nonspecific adsorption ofundesired biomolecules, and they should be highly specifictowards target miRNAs. Campuzano et al. developed an electro-chemical hybridization-based antifouling sensing platform fordetecting DNA sequences in human serum, which can be usedfor circulating miRNA.276 Suitable blocking agents such asbovine serum albumin, mercaptohexanol or poly(ethylene glycol)can be employed to avoid nonspecific binding.277



Tab

le1

Sum

mar

yo

fd

ete

ctio

nte

chn

olo

gie

so

fct

DN

A,

miR

NA

,C

TC

san

de

xoso

me

s

Bio

mar

ker

Tu

mor

type

Det

ecti

onpr

inci

ple

Met

hod

Mer

its

Lim

itat

ion

sR

ef.

ctD

NA

Bre

ast,

oste

o-sa

rcom

a,co

lore

ctal

,ov

aria

n

PCR

-bas

edA

RM

S/sc

orpi

onPC

RN

este

dre

al-t

ime

PCR

PCR

-SSC

PM

uta

nt

alle

le-s

peci

fic

PCR

Mas

ssp

ectr

omet

ryB

i-PA

P-A

ampl

ific

atio

nB

EA

Min

g

Low

cost

,ea

seof

use

Hig

hse

nsi

tivi

tySu

itab

lefo

rd

etec

tion

ofsp

ecif

icpo

int

mu

tati

ons,

copy

-nu

mbe

rva

riat

ion

s,sh

ort

ind

els

and

gen

efu

sion

sN

obi

oin

form

atic

san

alys

isD

etec

tion

ofct

DN

Aw

ith

very

low

mu

tan

tge

nes

Low

erse

nsi

tivi

ty,

can

only

det

ect

lim

ited

gen

omic

loci

Can

only

det

ect

lim

ited

gen

omic

loci

216

217

218

219

220

221

222

Bre

ast,

ovar

ian

,co

l-or

ecta

l,h

epat

ocel

-lu

lar,

pros

tate

,n

on-

smal

l-lu

ng

can

cer

Dig

ital

PCR

Dro

plet

-bas

edd

igit

alPC

RM

icro

flu

idic

dig

ital

PCR

PAR

E

Hig

hse

nsi

tivi

tyE

xpen

sive

223

224

and

225

226

Wh

ole-

gen

ome

sequ

enci

ng

Dig

ital

kary

otyp

ing

Wid

eap

plic

atio

nB

ioin

form

atic

sex

pert

ise

requ

ired

227

Tam

Seq

Safe

Seq

Doe

sn

otre

quir

epr

ior

know

led

geof

the

mol

ecu

lar

alte

rati

ons

Exp

ensi

ve22

822

9

Pan

crea

tic

du

ctal

aden

ocar

cin

oma,

gast

ric,

colo

n,

brea

st,

lun

g,co

lore

ctal

Tar

gete

dd

eep

sequ

enci

ng

Ion

-Am

pliS

eqT

MLo

wco

st,

hig

hse

nsi

tivi

ty23

0C

APP

-Seq

231

On

Tar

get

232

miR

NA

Pan

crea

tic,

oeso

-ph

agea

l,pr

osta

teN

orth

ern

blot

tin

gC

ombi

nat

ion

ofel

ectr

oph

oret

icse

para

tion

ona

mem

bran

efo

l-lo

wed

byh

ybri

dis

atio

nw

ith

labe

lled

prob

esan

dim

agin

g

Wid

ely

avai

labl

ean

dea

syto

perf

orm

Ted

iou

san

dti

me

con

sum

ing

233

and

234

Hig

hly

spec

ific

Bre

ast,

pros

tate

,ov

aria

n,

colo

rect

alga

stri

c,co

lon

,br

east

,lu

ng,

colo

rect

al

Mic

roar

ray

Hyb

rid

isat

ion

ofm

iRN

Aw

ith

com

plem

enta

rypr

obes

pre-

dep

osit

edon

tom

icro

arra

ypl

at-

form

spot

s

Est

abli

shed

prot

ocol

,pu

rpos

ebu

ilt

anal

ysis

tool

sLe

ssqu

anti

tati

ve,

qual

ity

miR

NA

ann

otat

ion

requ

ired

,u

nab

leto

dis

crim

inat

ecl

osel

yre

late

dm

iRN

Aeffi

cien

tly,

tim

eco

nsu

m-

ing,

poor

deg

ree

ofau

tom

atio

n,

low

sen

siti

vity

and

spec

ific

ity

235

and

236

Hig

hth

rou

ghpu

tsc

reen

ing,

sui-

tabl

efo

rd

isco

very

stu

die

s,in

expe

nsi

ve

Nex

t-ge

ner

atio

nse

quen

cin

gM

assi

vean

alys

isof

shor

tD

NA

sequ

ence

sin

para

llel

foll

owed

byse

quen

ceal

ign

men

tto

age

nom

eor

deno

vose

quen

ceas

sem

bly

Wid

esp

ectr

um

ofap

plic

atio

ns

Sen

siti

vean

dac

cura

teD

isti

ngu

ish

esm

iRN

Ava

rian

ts

Bio

info

rmat

ics

supp

ort

requ

ired

,w

ell

suit

edfo

rre

lati

vem

easu

re-

men

tson

ly

237

Poor

deg

ree

ofau

tom

atio

n

Bre

ast,

lun

g,co

lor-

ecta

l,ce

rvic

al,

gas-

tric

,pr

osta

te,

hea

dan

dn

eck

can

cer

qRT

-PC

RR

ever

setr

ansc

ript

ion

ofR

NA

tocD

NA

foll

owed

byqu

anti

tati

vePC

R

Wel

les

tabl

ish

edan

dw

idel

yu

sed

met

hod

Lim

ited

toh

igh

thro

ugh

put

scre

enin

gan

dd

isco

very

stu

die

s23

8an

d23

9W

ide

dyn

amic

ran

ge,

good

sen

siti

vity

and

spec

ific

ity

Eff

ecti

veon

lyag

ain

stes

tabl

ish

edR

NA

sSu

itab

lefo

rm

easu

rin

gsm

all

RN

Apa

nel

sC

ompa

tibl

ew

ith

labo

rato

ryw

orkf

low

s



Tab

le1

(co

nti

nu

ed)

Bio

mar

ker

Tu

mor

type

Det

ecti

onpr

inci

ple

Met

hod

Mer

its

Lim

itat

ion

sR

ef.

Ova

rian

,br

east

,pr

osta

te,

lun

g,ga

stri

c

Ele

ctro

chem

ical

Dir

ect/

indi

rect

adso

rpti

onof

miR

NA

onto

bare

gold

surf

ace

via

RN

A-g

old

affin

ity

inte

ract

ion

and

usin

gvo

ltam

met

ric

tech

niq

ues

tode

tect

the

resu

ltan

tcu

rren

tsi

gnal

s

Wel

lsu

ited

for

poin

t-of

-car

eas

says

base

don

pan

els

ofa

few

miR

NA

s

Com

plic

ated

and

mu

ltip

lese

nso

rfa

bric

atio

nst

eps,

tech

nol

ogy

not

read

yfo

rro

uti

ne

clin

ical

sett

ings

240–

242

Sen

siti

ve

Bre

ast,

gast

ric,

sali

-va

ryad

enoi

dcy

stic

carc

inom

a,th

yroi

d

Nan

ostr

ing

Mea

sure

sth

ebl

ocka

de

curr

ent

asa

resu

ltof

the

hal

ted

char

getr

ansp

ort

inth

en

anop

ore

byth

epr

esen

tm

iRN

Ata

rget

Hig

hly

sen

siti

vean

dex

cell

ent

dyn

amic

ran

geM

eth

odn

otav

aila

ble

ever

ywh

ere

Com

plic

ated

wor

kflo

w24

3an

d24

4

Bre

ast,

gast

ro-

inte

stin

al,

lun

g,co

lon

,co

lore

ctal

Imm

un

oass

ayT

wo

step

nu

clei

cac

idim

mu

-n

oass

ayw

ith

labe

lled

mon

oclo

nal

anti

bod

yto

DN

A/R

NA

het

eroh

ybri

ds

Suit

able

for

rou

tin

em

easu

re-

men

tsin

cen

tral

labo

rato

ryse

ttin

gs

Var

yin

gse

nsi

tivi

tyac

ross

diff

eren

tm

iRN

Apa

nel

san

dsa

mpl

ety

pes

245

Hig

hd

egre

eof

auto

mat

ion

Ora

l,br

east

,pr

ostr

ate

Surf

ace

plas

mon

reso

nan

ce(S

PR)

Mea

sure

sth

ere

frac

tive

ind

ex(R

I)ch

ange

sge

ner

ated

bysu

rfac

em

odif

ied

mol

ecu

lar

inte

ract

ion

sbe

twee

nR

NA

san

dth

ebi

orec

epto

r

Hig

hse

nsi

tivi

tyLo

wth

rou

ghpu

t19

2R

eal-t

ime

and

labe

l-fre

ean

alys

isN

otsu

itab

lefo

rro

uti

ne

mea

sure

men

ts

CT

CB

reas

t,co

lore

ctal

,lu

ng,

pros

tate

Cel

lSea

rch

Imm

un

omag

net

icen

rich

men

tm

eth

odA

nti

bod

y-co

ated

ferr

omag

net

icsp

her

es,

sem

iau

tom

atic

proc

ess

Cu

rren

tgo

ldst

and

ard

and

FDA

-ap

prov

edfo

rca

nce

rd

iagn

osis

,pr

ogn

osis

and

ther

apeu

tics

Sem

iau

tom

ated

syst

emSe

nsi

tive

Rep

rod

uci

ble

Hig

hpu

rity

Suit

able

for

mol

ecu

lar

prof

ilin

gan

dfl

owcy

tom

etry

Lim

ited

toC

TC

sw

ith

hig

hE

pCA

Mle

vels

Incr

ease

infa

lse

posi

tive

resu

lts

du

rin

gin

flam

mat

ory

con

dit

ion

sE

xpen

sive

inst

rum

enta

tion

Cos

tly

Mu

ltip

lest

eps

Non

-via

ble

cell

sM

odes

tce

llre

cove

ry

246

and

247

Pros

tate

,br

east

,co

lore

ctal

Mag

Swee

per

Imm

un

omag

net

icen

rich

men

tEp

CAM

-an

tibo

dyco

uple

dfe

rrof

luid

Non

-des

tru

ctiv

eca

ptu

rem

eth

odD

etec

tsE

pCA

M-p

osit

ive

CT

Cs

only

248

Au

tom

ated

Flex

ible

inpu

tsa

mpl

evo

lum

eIn

suffi

cien

td

ata

for

clin

ical

uti

lity

Hig

hpu

rity

Hig

hre

cove

ry

Pros

tate

,pa

ncr

eati

c,co

lon

Nan

ovel

cois

olat

ion

bysi

zeof

epit

hel

ial

tum

our

cell

s(I

SET

)

Nan

ovel

cosu

rfac

ew

hic

hal

low

ssi

ngl

etu

mou

rce

llca

ptu

reH

igh

sen

siti

vity

Sin

gle

step

sepa

rati

onpr

oced

ure

Pre-

proc

essi

ng

ofbl

ood

sam

ple

not

requ

ired

Low

cost

Lack

ofst

able

anti

bod

y-fu

nct

ion

alis

edch

ipFi

xed

cell

sD

etec

tson

lyE

pCA

M-p

osit

ive

CT

Cs

Insu

ffici

ent

dat

afo

rcl

inic

alap

plic

atio

n

249

Bre

ast,

cuta

neo

us

mel

anom

a,co

lor-

ecta

lca

rcin

oma

Mar

ker-

free

filt

rati

onap

proa

ch(s

ize)

EpC

AM

-pos

itiv

ean

dn

egat

ive

tum

our

cell

sar

ere

tain

edIn

her

ent

size

over

lap

betw

een

CT

Cs

and

WB

Cs

250

Sim

ple

proc

edu

reLo

wsp

ecif

icit

yA

vail

abil

ity

ofce

lls

for

add

itio

nal

stu

die

sFi

xed

CT

Cs

Req

uir

esan

auto

mat

edfi

ltra

tion

syst

emLi

mit

edd

ata

for

clin

ical

vali

dat

ion



Tab

le1

(co

nti

nu

ed)

Bio

mar

ker

Tu

mor

type

Det

ecti

onpr

inci

ple

Met

hod

Mer

its

Lim

itat

ion

sR

ef.

Pros

tate

,co

lon

,br

east

CT

C-c

hip

An

tibo

dy-

coat

edpi

ns

insi

de

the

chip

,bl

ood

flow

sbe

twee

nth

epi

ns

Hig

hse

nsi

tivi

tyC

ells

not

suit

able

for

tiss

uecu

ltur

e25

1N

obl

ood

sam

ple

pre-

proc

essi

ng

requ

ired

EpC

AM

-pos

itiv

eC

TC

sca

non

lybe

det

ecte

dLo

wsa

mpl

ein

put

Prot

otyp

eC

anbe

appl

ied

for

mol

ecu

lar

prof

ilin

gC

ell

viab

ilit

yis

mai

nta

ined

Spec

ific

Hig

hd

etec

tion

rate

(app

roxi

-m

atel

y10

0%)

Lun

g,br

east

,pr

os-

tate

,h

ead

and

nec

kca

nce

r

CT

C-i

Ch

ipH

ydro

dyn

amic

sort

ing,

iner

tial

focu

sin

gM

arke

r-fr

eeis

olat

ion

252

Bre

ast,

ovar

ian

Flow

frac

tion

atio

nco

upl

edw

ith

die

lect

roph

ores

isSe

para

tion

base

don

tota

lce

llca

paci

tan

ceV

aria

tion

sin

cyto

plas

mic

and

mem

bran

eco

nd

uct

ivit

yov

erti

me

253

and

254

Bre

ast,

skin

,n

on-

smal

lce

lllu

ng

can

cer

Invi

voC

TC

det

ecto

rM

edic

alSe

ldin

ger

guid

ewir

efu

nct

ion

alis

edw

ith

EpC

AM

inse

rted

intr

aven

ousl

y

Rea

lti

me

mon

itor

ing

No

bloo

dpr

oces

sin

gre

quir

ed25

5an

d25

6

Pros

tate

Ele

ctro

chem

ical

det

ecti

onch

ipco

upl

edw

ith

capt

ure

circ

uit

Cir

cuit

for

CT

Cca

ptu

rein

tegr

ated

wit

hly

sis

and

mR

NA

anal

ysis

Com

bin

edca

ptu

rean

dan

alys

ism

inim

ise

sam

ple

han

dli

ng

257

Bre

ast,

pros

tate

EPI

SPO

TD

eple

tion

ofC

D45

+ce

lls

Det

ects

viab

leep

ith

elia

lse

cret

ing

cell

s18

3

Pan

crea

tic,

non

-sm

all

cell

lun

gca

nce

r

Apt

amer

-mod

ifie

dm

etal

nan

owir

esA

ptam

er-b

ased

capt

ure

ofC

TC

sU

nbi

ased

enri

chm

ent

ind

epen

-d

ent

ofC

TC

/DT

Cph

enot

ype

Low

stab

ilit

yof

apta

mer

sin

bloo

d25

8

Bre

ast,

pros

tate

,m

elan

oma

Clu

ster

-Ch

ipU

ses

bifu

rcat

ing

trap

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7.1.3 Varying sizes. Circulating DNA was among the firstbiomarkers examined for cancer staging with the increasedserum DNA concentrations associated with metastatic cancerand other related conditions, including sepsis and autoim-mune disease.278 Circulating tumor DNA accounts for a smallerportion of total cfDNA, especially in early stage cancer andminimal residual conditions, and thus its detection in plasmais difficult.279 High fragmentation of cfDNA represents oneof the major concerns in determining circulating DNA. DNAfragmentation decreases intact or analyzable DNA copiescontaining genetic markers of interest, thus causing analysissuch as sequencing and amplification difficult.2 More so,isolation and precise quantification of circulating DNA areaffected by the potential loss of DNA characteristics beingexploited in the designed analysis.

The length of RNA ranges between 18 and 20 nt for smallRNA such as miRNA and 4200 nt for large RNA such as longnon-coding RNA (LncRNA). Typically, it is difficult to detectshort RNAs using amplification-based methods due to thepossible size match with selected primers. To circumvent thischallenge, short RNA can be polyadenylated enzymaticallyresulting in longer sequences, which can be used in the reversetranscription step in RT-qPCR driven by oligo-dT primers.280 Astem-loop primer can also be used during reverse transcriptionto form a nicked RNA hybrid via hybridization with the 30 endof the target RNA sequence.281 Conversely, detection of longRNAs based on hybridization techniques poses significantchallenges due to the possible formation of secondary andtertiary structures on the sensor platform.282,283

7.1.4 Difficulties in direct analysis. The commonly usedmethods for nucleic acid analysis are based on amplificationand hybridization. Hybridization provides sequences with highspecificity suitable for cancer specific molecular alteration-based ctDNA analysis. However, longer processing times andmulti-steps are incurred during the denaturation of doublestranded DNA. In addition, renaturation of ssDNA also inhibitsthe efficiency of hybridization-based analyses.284 Variouselectrochemical methods based on DNA clutch probes andalkaline phosphatase labelled Zn-finger proteins have beenproposed to overcome this challenge.284,285 In the clutch probeapproach, Shana O Kelley and colleagues have used DNA clutchprobes (DCPs) for detecting ctDNA with high sensitivity andspecificity. DCPs enable efficient hybridization analysis bypreventing renaturation of DNA molecules.274 The assay enabledthe detection of 1 fg mL�1 of target mutation in the presence of100 pg mL�1 of wild-type DNA (Fig. 11).285

7.1.5 Low stability. At room temperature, RNA is normallyunstable because of possible degradation by ribonucleases(RNases). Henceforth, RNases can affect the precise detectionof RNA through continual degradation during incubation steps.To overcome this hurdle, Frie et al. highlighted the incorpora-tion of RNase inhibitors into the assay.286 In this approach,cells are permeabilized in the presence of RNAse inhibitors thatinhibit endogenous and exogenous RNAses.

Different forms of circulating DNA display different levels ofstability.287,288 The process of release, degradation and clearance

of cfDNA involves several steps. The varying levels of DNaseactivity and attachment of cfDNA on the blood cell surface affectthe clearance time and their stability.289,290 Various mechanismsand organs are associated with cfDNA clearance, uptake anddegradation by phagocytes.291,292 As a result, quantifying theamount of cfDNA in diagnostic approaches presents inconsistentand insufficient results.293

7.1.6 Similar sequences. The high similarity of miRNAsequences together with their short length often poses furtherdifficulties for isolating them. Micro RNAs from the samefamily can have different substantial outcomes in the body;for example, let-7a and let-7c which differ in the sequence by asingle nucleotide are decreased in lung and breast cancerrespectively.8,294 Therefore, accurate detection and quantificationof specific miRNA may be influenced by nucleotide base-pairmismatch. Highly specific recognition systems are required todetermine sequences that differ only by a single nucleotide.Recently, the use of locked nucleic acid (LNA) probes has emergedas a potential strategy to measure similar target sequences.When encapsulated into DNA probes, LNA analogues increasethe melting temperature which enhances the binding affinity andsensitivity up to 10-fold.295 More so, further complication maybe incurred when using hybridization-based methods to com-pare sequence heterogeneity between the genomic precursorsand 30 and 50 ends of isomiRNAs.296,297

7.2 Technical challenges of CBs

The need for non-invasive molecular profiling tools in recentyears has grown substantially due to the increasing need andbetter understanding of genomic alterations and personalizedtreatment options. The integration and utilization of CBs inroutine clinical settings is of utmost importance. Numerousvalidation studies are required to provide substantial evidenceof the efficiency and reliability of the markers for the clinicalutility of the developed tests. To establish the cancer definingvariants, cancer-related biomarker profiles of large numbers ofcontrols and healthy individuals are required, and continuousfollow-ups are needed to identify false positive signals.2 Currentdemonstrations often face technical challenges associated withpreanalytical conditions that need to be standardized such assample collection and processing.

7.2.1 Sample preparation and choice of the sample source.The choice of the source for CBs particularly miRNA may have aprofound effect on the sequence expression profile obtained.MiRNAs have been found to exist in numerous biological fluidsand therefore it is imperative to understand the sources ofmiRNAs that are significant for clinical use. Currently, circulat-ing miRNAs are extracted from venous plasma or serum with agood correlation between their expression levels.298 It has beenobserved that miRNAs obtained from plasma and serum werenot affected by multistep treatments such as freeze–thaw cycles,boiling, long-term storage or high pH.299 However, duringplasma preparation, the cellular pellet is susceptible to con-tamination when pipetting away the supernatant. The presenceof sizeable inter-sample flexibility of protein and lipid inindividual serum or plasma samples could inhibit the impact



of the RNA extraction efficiency and potentially presentpossible inhibitors to qRT-PCR. Furthermore, it is noted thatplatelets and red blood cells also bear disease-specific miRNAs,which may result in contrived high miRNA concentrations as aresult of hemolysis or improper processing of the bloodsample.8 Vesicles/exosomes are also known to be carriersof circulating miRNA. Recent studies have indicated thatexosomes obtained from serum and saliva are highly enrichedin miRNAs and most of these miRNAs are enclosed in micro-vesicles, primarily exosomes, and do not circulate freely inhuman biological fluids.300,301 Thus, exosomal miRNA can beapplied in preliminary biomarker selection studies to overcomethe occurrence of false negatives by low concentrated miRNAthat may be omitted by using unfractionated serum or saliva.300

7.2.2 Inconsistency of different isolation methods. Commer-cially available protocols for isolating miRNAs from serum andplasma are based on liquid–liquid extraction such as guanidium-phenol extraction preceded by isopropanol precipitation ofmiRNA in aqueous phase.302 Column-based methods such asmiRNeasy (Qiagen) and mirVana (Invitrogent AM1560) are alsoused. However, it is difficult to achieve consistency owing tothe short sequences and low concentrations. In addition, theapplication of pre-amplification reagents in some studies to

enhance qRT-PCR sensitivity requires standardization. Smalldiscrepancies in centrifugation rates in various protocols mayhave a significant effect on the isolation efficiency of particulatesof interest resulting in extra variables added to extracted miRNA.

In the case of exosomes, one of the major drawbacks ofroutine diagnostic application of exosomes as cancer biomar-kers is the method of isolation used. Presently, there is noestablished gold standard method for exosome isolation.303,304

Owing to their low density and small size, exosomes arenormally isolated from cell culture media and body fluids bydifferential ultracentrifugation.305 Although ultracentrifugationis shown to be reproducible and yield optimal amounts ofexosomes, it is labor-intensive, time consuming and requiresexpensive equipment, thus limiting its clinical applications inresource-poor settings. In addition, exosomes are subjected toextreme pressure during processing which can result in thereduction of highly specific exosomes during precipitation.306

Furthermore, it is difficult to reproduce exactly the isolation indifferent settings or places. Another isolation method fre-quently used is ultrafiltration which is based on size exclusion.Exosomes are separated based on their size using membranefilters with defined molecular weight or size exclusion limits.102

Compared to ultracentrifugation, ultrafiltration is relatively fast

Fig. 11 Schematic representation of the clutch probe approach for ctDNA detection. (A) Detection approach. Double stranded DNA is denatured togenerate ssDNA, and DNA clutch probes are then introduced to inhibit the renaturation of ssDNA. The PNA clump hybridizes with the perfectly matchedwild type target ssDNA, whereas the mutant target ssDNA remains unhybridized. (B) Detection technique based on the chip. Nanostructuredmicroelectrodes (NME) are immobilized with PNA probes complementary to mutant target DNA and only complementary mutant targets bind to theprobe. The resultant signal generated is recorded in the presence of a reporter system. (C) Sensor design. The sensors are deposited in arrays on themicrochip with the inset showing the cross-sectional hole in the chip. (D) An image showing the NME sensor using scanning electron microscopy.Reprinted with permission from ref. 285. Copyright 2016, American Chemical Society.



with no special equipment required.306 However, the applica-tion of force may give rise to deformed or breaking up ofvesicles which makes it difficult to use them in downstreamapplications. This is especially in the analysis that requires ahigh exosome concentration, such as the determination of theirmiRNA profile.103 Recently, many commercial kits compatiblewith body fluids based on precipitation have been developed.306

Their reproducibility and rapidity make them useful for futurediagnosis, especially in miRNA-based tests. The emergence ofmicrofabrication technology has presented a unique opportunityin microfluidics-based assays by probing the physical and bio-chemical characteristics of exosomes at microscales. Acoustic,electrophoretic and electromagnetic manipulations can also beused as innovative sorting mechanisms for exosomes.114,176,307

Advantages of using these devices include the reduced samplevolume, reagent consumption and isolation time.

7.2.3 Specificity challengesCTCs. Different subsets of CTCs can exhibit various pheno-

types during EMT. Thus, a desirable broad-enrichment spec-trum based on the application of a specific combination of cellsurface epithelial and mesenchymal markers is needed.308

However, a vast combination of disparate markers encompass-ing all CTC phenotypes may enhance the chances of single cellsexpressing one of these markers leading to false positive results.To overcome this challenge, scientists have often targeted actinbundling protein plastin 3, which is not expressed in blood cellsand also not downregulated by CTCs during their EMT. Recently,microfluidic channels coalesced with surface cell markers orphysical properties, such as IsoFlux,309 are extensively appliedin CTC enrichment strategies, and substantially enhance effi-cient and accurate CTC analysis.310,311 Furthermore, Karabacaket al.312 described an isolation procedure of CTCs usingCTC-iChip technology that integrates cell surface markers andphysical properties. This system showed promising results,recovering more CTCs with less nucleic acid damage. AfterCTC enrichment, some CTCs may be retained as backgroundcells, causing low efficiency for analysis and sequencing.313

However, recent advancements in next generation sequencing(NGS) and single-cell-sequencing technologies allow for theimplementation of techniques such as flow cytometry analysis(FACS) and laser capture microdissection (LCM). These furtherisolate CTCs from the background cell pool.

Furthermore, tumor specific markers such as mammaglobinfor breast cancer and prostate-specific antigen (PSA) forprostate cancer, are specific but can be downregulated duringdedifferentiation of tumor cells.314–316 Another challenge asso-ciated with CTC detection is the limited supply of bloodsamples from available cancer patients. This may foist seriousdifficulties when using micro-devices to detect rare CTCs inearly stage cancer, in which the CTC count levels are relativelylow. To alleviate this challenge, one may use a Cell Collectordirectly to collect CTCs. Patient’s blood is passed through afunctionalized area of the collector, which filters it and enablesbinding of CTCs by antibodies against EpCAM.313 Leukapheresisis another clinical approach that is commonly employed forisolating mononuclear cells from large amounts of blood.

Leukapheresis uses flow cytometry and RT-PCR to analyzeCTCs via elutriation.317 Recently, leukapheresis has beenused on non-metastatic cancer patients for molecular analysiswhere it enabled the determination of about 100 000 CTCs perpatient and established a correlation of CTC numbers withdisease spread.318

CtDNA. Several preanalytical factors such as blood collection,delay in processing, centrifugation protocols, sample shipment,storage conditions, choice of anticoagulant, etc. can affect theresults of CB analysis.319–322 Delays between sample collectionand isolation of plasma can affect the intact DNA as a resultof peripheral blood cell lysis which subsequently affects thePCR and tagmentation-based sequencing strategies by reducingthe available effective template for analysis.323 Thus, there is aneed for special cell-stabilization collection tubes that have thecapacity to hold a preservative that can inhibit the lysis ofperipheral blood cells for many days at room temperature. Theability to perform centrifugation in various centrifuges or storingat 4 1C after collection for a short period of time, will markedlyimprove the possibility of collecting high quality samples.Previous reports predominantly focused on PCR or dPCR basedsingle locus-specific analysis. However, the use of the multi-plexed droplet digital PCR strategy to examine amplifiable ctDNAand evaluate large molecular weight background DNA in a singlestep exhibits a greater resolution and large genomic coveragethan a locus-specific approach.279,323 Multiplexing techniquesplay a critical role in sequencing experiments by aiding inthe optimization of sample input to attain reproducibleperformances. Several regions in the genome are targeted, thusminimizing input DNA required for quality assessment. Locus-specific amplification bias or somatic copy number changesoften found in qPCR or digital PCR assays designed for singleor double loci targets are also avoided.323

Notwithstanding the recent advancements and improve-ments in designing isolation and purification protocols, size-able sample amounts are still lost during the purification stage.Previous studies have shown that subjecting samples to highcentrifugation speeds results in ex vivo release of DNA fromblood cells.324 However, the incorporation of microfluidics andnanotechnology has opened new avenues for cfDNA isolationthat can avoid tedious purification steps. Campos et al. con-structed a polymer-based microfluidic device for extraction ofcfDNA. The device enabled efficient cfDNA/ctDNA extractiondirectly from plasma.325

7.2.4 Low sensitivity. The concentration of clinically relevantCBs in tissues, serum and other body fluids is relatively low andthus, sensitive and specific molecular profiling strategies needto be designed for their extraction. In the case of ctDNA, variousnew extraction methods have recently been designed includingmagnetic-bead based techniques, organic solvent-basedisolation and commercial kits.326–330 Following the earliestdiscovery of ctDNA in plasma by Mandel and Matias, theycould isolate about 5.4 mg mL�1 total DNA in the plasma ofhealthy individuals.331 In addition, the current column-basedmethods have demonstrated higher sensitivity compared to the



previously used protocols such as the salting-out DNA isolationprotocol.332

8. Conclusions and futureperspectives

To date, conventional tissue biopsy for pathological diagnosis ofcancer is immensely invasive and general imaging screeningssuch as tomography, scans and magnetic resonance imagingscans are expensive and scarce in many clinics. Thus, blood-based assays for screening and identifying tumor relatedmarkers are of great importance and can significantly reducehealthcare costs. In this review, we have provided an overviewof CBs (ctDNA, miRNA, CTCs and exosomes) as non-invasivecancer biomarkers with applications in diagnosis, prognosisand therapy response and the recent developments in theirdetection technologies. We have also articulated the mainmethodological deficiencies of some of these technologies.NGS approaches have the ability to avoid limitations associatedwith PCR amplification such as the error rate for DNA poly-merase which limits the application of PCR-based methods thatrely on sensitive amplification. Furthermore, there is need forimproved analytical and diagnostic assays that are sensitivetowards genome-wide ctDNA because of the lack of recurrentgenetic alterations by many tumor types which can revealspecific cancer markers. In addition, more attention needs to bedevoted towards intratumoral heterogeneity.333 Since it is notclear whether ctDNA constitutes DNA from well-defined sub-clones, more interrogations need to be conducted with respectto clinical, molecular and imaging studies to assess clinicalprogression and therapeutic resistance. Several attempts havebeen made towards the development of reliable assays for tumorgenomes derived from ctDNA. Most laboratory procedures aretedious and costly which limits the actual application in adiagnostic setting. However, future advancements in sequencingtechnologies and chip designs are believed to have a potential toanalyze genome-wide ctDNA with high sensitivity at a lower cost.

We have also evaluated the current progress and limitationsin miRNA-detection technologies together with their clinicalrelevance in diagnosis and prognosis. It has been establishedthat miRNA can operate as a liquid-biopsy marker for noninva-sive diagnosis of cancer. Among the detection technologiesof miRNA, electrochemical based assays generally entail thehybridization of target miRNA with a complementary captureprobe followed by an electrochemical readout method. How-ever, in spite of the huge progress made in electrochemicalstrategies, extensive work is still required to integrate them intoportable point-of-care devices. Generally, many electrochemicalbiosensors are solely proof-of-concept demonstrations whichrely mostly on complicated fabrication steps of the sensor and aseries of optimizations in a well-equipped centralized facility.Hence, there are many challenges involved in order to translo-cate these laboratory-based proof-of-concept demonstrations toclinical applications. One of the challenges is the false-positivedetection of miRNA as a result of nonspecific binding.

Nanomaterials have also been incorporated for the construc-tion of miRNA biosensors to amplify and obtain sensitivedetection signal. Since highly efficient technologies for miRNAcontinue to develop, we anticipate that an ideal miRNA sensorwith desired clinical applications will be constructed in thenear future.

The deficiency of technologies that can effectively isolateand detect CTCs poses a major challenge to the understandingand application of CTC biomarkers in clinical settings. Theheterogeneity of biomarkers currently employed for capture orenrichment of CTCs and the notable bias of currently availableplatforms are the key to technological limitations of CTCswhich require more attention. Despite the progress shown inCTC detection technologies,259 more time is required to translatethese advances to routine clinical applications. For more than adecade, the CellSearch system still remains the only FDAapproved CTC based cancer diagnosis system and has not beenimplemented into routine clinical practice due to insufficientlyproven clinical merits,334 in spite of the continuous evolution ofthe technology.335 Therefore, more focus should be directedtowards detailed molecular and functional characterization ofsingle CTCs. High throughput analytical techniques such as NGShave enabled the analysis of whole genomes and transcriptomesof single CTCs, and with the possibility of automation, large CTCmolecular landscapes have been determined.336–338

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the Australian Research CouncilDiscovery Projects (DP190102944 and DP180100055), Australia-Korea Foundation (Department of Foreign Affairs and TradeGrant, AKF2018043), and HDR Scholarship from GriffithUniversity. We also acknowledge Surasak Kasetsirikul for thecover art of the article.

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