programmable bio-nano-chip systems for serum ca125...

Research Article

Programmable Bio-Nano-Chip Systems for Serum CA125Quantification: Toward Ovarian Cancer Diagnostics at thePoint-of-Care

Archana Raamanathan1,4, Glennon W. Simmons1, Nicolaos Christodoulides1, Pierre N. Floriano1,Wieslaw B. Furmaga5, Spencer W. Redding6, Karen H. Lu2, Robert C. Bast Jr3, and John T. McDevitt1

AbstractPoint-of-care (POC) implementation of early detection and screening methodologies for ovarian cancer

may enable improved survival rates through early intervention. Current laboratory-confined immunoa-

nalyzers have long turnaround times and are often incompatible with multiplexing and POC implemen-

tation. Rapid, sensitive, and multiplexable POC diagnostic platforms compatible with promising early

detection approaches for ovarian cancer are needed. To this end, we report the adaptation of the

programmable bio-nano-chip (p-BNC), an integrated, microfluidic, and modular (programmable) plat-

form for CA125 serum quantitation, a biomarker prominently implicated in multimodal and multimarker

screening approaches. In the p-BNCs, CA125 from diseased sera (Bio) is sequestered and assessed with a

fluorescence-based sandwich immunoassay, completed in the nano-nets (Nano) of sensitized agarose

microbeads localized in individually addressable wells (Chip), housed in amicrofluidicmodule, capable of

integratingmultiple sample, reagent and biowaste processing, and handling steps. Antibody pairs that bind

to distinct epitopes on CA125 were screened. To permit efficient biomarker sequestration in a three-

dimensional microfluidic environment, the p-BNC operating variables (incubation times, flow rates, and

reagent concentrations) were tuned to deliver optimal analytical performance under 45minutes.With short

analysis times, competitive analytical performance (inter- and intra-assay precision of 1.2% and 1.9% and

limit of detection of 1.0 U/mL) was achieved on this minisensor ensemble. Furthermore, validation with

sera of patients with ovarian cancer (n ¼ 20) showed excellent correlation (R2 ¼ 0.97) with gold-standard

ELISA. Building on the integration capabilities of novel microfluidic systems programmed for ovarian

cancer, the rapid, precise, and sensitiveminiaturized p-BNC system shows strong promise for ovarian cancer

diagnostics. Cancer Prev Res; 5(5); 706–16. �2012 AACR.

IntroductionIn 2010, approximately 21,880 women will be diag-

nosed with ovarian cancer in the United States and13,850 women will succumb to this disease (1). Although90% of ovarian cancers can be treated at stage I with thecurrently existing surgical and chemotherapeutic regi-mens, only 25% of ovarian cancers are detected at this

treatable stage due to the nonspecific symptoms and thelack of effective screening procedures (2–4). Giventhe low prevalence of ovarian cancer (1 in 2,500 post-menopausal women), an extremely high specificity(99.6%) and sensitivity (>75%) are required to achievea minimum positive predictive value (PPV) of 10% (i.e.,10 laparotomies/case of ovarian cancer detected; ref. 5).No screening test exists currently for recommended usein the general population, underscoring the need fornovel early detection and easily completed screeningmethods (6).

For suspected pelvic masses, ovarian cancer diagnosis isrealized by pelvic examination, transvaginal sonography(TVS), and serum CA125 leading to exploratory or diag-nostic laparoscopy (4). TVS provides a precise image of theovary andwhereas PPVs in themost promising studies havebeen reported to be close to 10%, prohibitively high costsfor implementation has precluded its use as a first-linescreen (2, 7, 8).

The biomarker CA125 is a heavily glycosylated, high-molecular-weight protein, encoded by the MUC16 gene,with a potential role in contact and adhesion for

Authors' Affiliations: 1Departments of Bioengineering and Chemistry,Rice University; Departments of 2Gynecologic Oncology and 3Experimen-tal Therapeutics, The University of Texas MD Anderson Cancer Center,Houston; 4Departments of Chemistry and Biochemistry, The University ofTexas at Austin, Austin; Departments of 5Pathology and 6Dental DiagnosticScience, University of Texas Health Science Center at San Antonio, SanAntonio, Texas.

Note:Supplementary data for this article are available atCancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

Corresponding Author: John T. McDevitt, Departments of Bioengi-neering and Chemistry, Rice University, 6100 Main Street, MS 142,Houston, TX 77005. Phone: 713-348-2123; Fax: 713-348-2302; E-mail:[email protected]

doi: 10.1158/1940-6207.CAPR-11-0508

�2012 American Association for Cancer Research.

CancerPreventionResearch

Cancer Prev Res; 5(5) May 2012706

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Published OnlineFirst April 9, 2012; DOI: 10.1158/1940-6207.CAPR-11-0508

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metastasizing epithelial ovarian cancer cells (9, 10). Theextracellular domain shed in serum from the surface of theovarian cancer cells following cytoplasmic phosphorylationand proteolytic cleavage is elevated in 80% of advanced-stage carcinomas (11, 12). Hence, serum CA125 has beenextensively used and has been U.S. Food and Drug Admin-istration (FDA)-approved for recurrent disease detectionand monitoring chemotherapy response (13). However,CA125 is elevated in only 50% to 60%of early-stage cancerswith false-positives for a variety of nonmalignant gyneco-logic and physiologic conditions (14, 15).The inaccessibility of the ovaries for screening has

generated extensive interest in noninvasive serum biomark-er–basedmethodologies for first-line screens and 2 approa-ches are being investigated. In multimodal screening, thepatient’s risk for ovarian cancer is stratified by monitoringlongitudinal values of CA125, interpreted with a Risk ofOvarianCancer Algorithm (16, 17).On the basis of assessedrisk levels, patients are triaged for follow-up with CA125,TVS, or surgery (18). In an ongoing, large, randomizedcontrolled trial of 202,638 women (UK Collaborative Trialof Ovarian Cancer Screening; UKCTOCS), the multimodalscreening (MMS) showed promising PPVs of 43.3% (19).Nevertheless, 20% of ovarian cancers do not express CA125and in multimarker panel (MMP) screening, multiple bio-markers taken together as a panel, have shown improvedsensitivity and specificity compared with CA125 alone(2, 20, 21). Such biomarkers have been identified withproteomic techniques (22, 23), with the most promisingpanels comprising 3 to 4 biomarkers, presented as comple-mentary to CA125 (24, 25).The point-of-care (POC) implementation of aforemen-

tioned biomarker-based early detection and screeningmethodologies may potentially impact survival ratesthrough early intervention with increased access to rapid,low-cost, and large-scale population screening. Alignedwith promising research, such diagnostic platforms pro-grammed for ovarian cancer should entail multiplexability,high-quality analytical performance (precision and sensi-tivity), and sample economy. Multiplexability and sampleeconomy are crucial for validation of MMPs discovered byproteomic techniques with limited supply of early-stagepreclinical samples (26, 27). Furthermore, longitudinalmonitoring of CA125 and other biomarker levels necessi-tate high precision to discern biologic variations and lowlimit of detection (LOD) to facilitate early disease detection(28). Finally, rapid analysis times will permit immediateaccess to TVS,when required andminimize follow-up visits,with additional advantages of reduced patient discomfortand anxiety. Amultifunctional diagnostic platform that canbe used for assay development and validation and in turncan function as the endpoint diagnostic will significantlyreduce residence time in the "diagnostic pipeline para-digm," enabling speedier translation of biomarkers fromdiscovery to diagnostics (23). Current gold-standard meth-odologies such as ELISA, dependent on centralized labora-tory infrastructure, have typical turnaround times on theorder of 24 to 48hours (29). Furthermore, the singlemarker

at a time approach and the need for trained personnel areinconducive for multiplexing and POC implementation(29, 30). While flow cytometric bead–based approachestackle the issue of multiplexing and have served well forbiomarker discovery, other technological constraintsremain similar (31).

Microfluidic platforms have been proposed for POCapplications in cancer diagnostics due to their inherentadvantages of low cost, sample and reagent economy, lowLODs, rapid analysis times, portability, automation, andease of operation by nonexpert users (29). However, torealize widespread distribution, highly application-orient-ed systems tailored to address specific analytical andresearch needs of the pertinent clinical community (asdiscussed earlier for ovarian cancer) are needed (32).Unfor-tunately, technological maturation of microfluidic plat-forms has not complemented advances in cancer biomarkerdiscovery attributable to the apparent disconnect betweenthe relevant communities, crucially important for this high-ly interdisciplinary translational effort (33). In terms oftechnological constraints, reliance on centralized laborato-ry infrastructure akin to conventional immunoanalyzersneeds to be overcome through miniaturized "analyzer"components to supplement the microfluidic device (34).Although an instrument-free setup would be preferred,high-sensitivity quantitative analysis for ovarian cancerbiomarkers necessitates optical detection and consequent-ly, miniaturized optical and mechanical components forPOC implementation (35). Following sample input, allsubsequent processes need to be automated with minimaluser interaction in a closed format suitable for biohazard-ous material handling (29).

Rapid, reliable, and efficient measurement of multiplekey biomarkers simultaneously at the POChas the potentialto transform clinical laboratory science. Toward this goal,the McDevitt laboratory has sustained efforts to improvePOC in vitro diagnostics through the development, valida-tion, and implementation of programmable Bio-Nano-Chips (p-BNC; refs. 36, 37). The programmable feature ofthese systems refers to the capacity of the sensor ensemble tofunction as a standard platform that can be retasked (i.e.,programmed) to serve a new application through insertionof molecular level code (i.e., the biomarker-specificreagents). The Bio terminology relates to the capacity tomeasure and extract the biosignatures associated with dis-ease progression. The Nano element describes the capacitytominiaturize the system embodied in the use of nano-netsfor efficient and rapidbiomarker capture aswell as quantumdots for increased signal generation. The Chip term empha-sizes the capacity to mass-produce the sensor elements inways analogous to those used by the microelectronicsindustry. In this work, we report our initial efforts tocustomize the p-BNC system, to address specific needs inovarian cancer biomarker–based screening, using CA125 asthe proof-of-concept biomarker. Here, we explore criticalvariables pertaining to reagent screening and assay devel-opment in a microfluidic environment that serve to influ-ence the analytical performanceof theminisensor ensemble

Programmable Bio-Nano-Chip for Ovarian Cancer Diagnostics

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under time constraints imposed for POC applications.This report further details CA125 quantitation in sera ofpatients with advanced-stage ovarian cancer and validationagainst gold-standard platforms and explores the potentialof the p-BNC system in the context of MMS and MMPscreening strategies.

Materials and MethodsImmunoreagents and buffers

All reagents used for conducting immunoassays in the p-BNCs were prepared in PBS (Thermo Fisher Scientific)containing 0.1% (w/v) bovine serum albumin (BSA; Sig-ma-Aldrich) needed for reagent stability and blocking non-specific binding (PBSA). Anti-CA125 monoclonal antibo-dies (Clones M002201, M002203, M77161, M8072320,M8072321, andM8072322) were obtained from FitzgeraldIndustries International. Low cross-reactivity, calibratorgrade CA125 standards were acquired from Meridian LifeScience. Human serum–based Liquicheck Tumor Markercontrols were procured from Bio-Rad for precision studies.

p-BNC construction and assay executionThe various generations of p-BNCs were fabricated using

xurography-based rapid prototyping techniques. Technicaldetails about construction are provided in the Supplemen-tary Information section.

A typical assay was executed with analyte-specific beadsand negative control beads [interleukin (IL)-6] localized inthe individually addressablewells on the chip housed in thep-BNC microfluidic ensemble. Alternatively, beads of vary-ing concentrations or antibody clones were multiplexedalong adjoining columns and identified by location foroptimization studies. Following a 1-minute high flow ratestep (2,500 mL/min) for priming and microbubble elimi-nation, the serially diluted analyte of predetermined con-centration in PBSA or the serum sample was introduced tothe bead array. Any remaining uncaptured analyte follow-ing this step was removed with a high-stringency, high flowrate PBS wash. The immunosandwich was completed in theagarose nano-nets with the introduction of Alexa Fluor488–conjugated detecting antibody, followed by removalof excess antibody with a high-stringency wash and digitalimage capture of the fluorescent beads to quantify thecaptured analyte.

CA125 ELISAELISAs were conducted at MD Anderson Cancer Center

(MDACC; Houston, TX) and University of Texas, HeathScience Center at San Antonio (UTHSCSA; San Antonio,TX) with standard manufacturer protocols (Elecsys CA125II electrochemiluminescence assay on the "cobas e" immu-noanalyzer; Roche diagnostics).

Sample collection and analysisSerum samples were collected from patients with

advanced-stage (III and IV) ovarian cancer with informedconsent following Institutional Review Board–approvedprocedures at the MDACC (n ¼ 10) and UTHSCSA (n ¼

10) using routine protocols (See Supplementary Infor-mation). The aliquoted samples were analyzed by ELISAat the collection institutions and sent to the McDevittlaboratory over dry ice for analysis on the p-BNCs.

Image analysisImages obtained were analyzed using open-source Ima-

geJ (NIH, Bethesda, MD) software with custom imageanalysis macros to yield the mean fluorescence intensity(MFI) for the corresponding area of interest on the beads(38). Further details are provided in the Supplementarysection.

Statistical analysisCalibration curves were generated with SigmaPlot 10

(SPSS Inc.) and fitted to a 4-parameter logistic equation.Linear regression, determination of unknown concentra-tions from standard curves, and curve fitting were con-ducted using the same software. Precision analysis wasconducted via the methods analysis module of MicrosoftExcel. All optimization experiments were carried out intriplicate and error bars denote interassay precision unlessotherwise noted.

Resultsp-BNC systems at the point-of-care

The p-BNC system (Fig. 1) is a highly sensitive, multi-plexable, POC-amenable microfluidic platform consis-tent with requirements set forth for the next generationof ovarian cancer diagnostic devices (see Introduction).This minisensor ensemble consists of 2 main parts: adisposable cartridge that contains the programmable chipmodule and portable instrumentation that serves as theuser interface, thereby reading, analyzing, storing data,and reporting the results. This system is suitable for point-of-service quantitation of CA125 and expanded multi-plexed panels that may be completed with finger-stickquantities of blood.

The completely integrated p-BNC (Fig. 2A, III) is com-posed of modular subassemblies, built around a micro-machined stainless steel bead holder chip housing theprogrammable agarose microbead "immunoanalyzer"core, sandwiched in a plastic microfluidic card, that inte-grates various on-card fluid handling processes, a sampleinput port, and reagent containing blister packs. The pro-grammable microbead immunoanalyzer core is identicalbetween various generations of the p-BNCs (Fig. 2A, I, III)and is capable of high-efficiency analyte capture in a3-dimensional (3D) agarose nano-net (Fig. 2B, III), seques-tered from biologic matrices to aid rapid, ultrasensitivequantitative measurements of disease biomarkers in a fluo-rescence-based sandwich immunoassay format (Fig. 2C,I–IV). The integrated microfluidic assemblage contains asample loop with an overflow chamber for precise on-cardsample metering, in-line filters, and selectively permeablemembranes for debris and bubble removal, reagent pads,and blister packs for on-card reagent and buffer storage, in-line micromixers for uniform reagent delivery to the

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immunoanalyzer core, and an on-card waste disposalchamber (Fig. 2A, III). Efforts from our translational part-ners have led to the development of aminiaturized analyzer(Fig. 1) with scaled-down light-emitting diode (LED)-based

optics, a miniaturized microscope and camera, battery-powered source, and linear actuated steppermotors to crushthe blister packs and to release buffer and reagents to drivethe immunoassay to completion, followed by optical

Sample meter Immunoanalyzer

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Figure 1. Illustration of the envisioned POC use of the p-BNCs for early detection and screening of ovarian cancer. Sample is obtained from the patient viavenipuncture (serum) or finger-stick (left) and transferred to the p-BNC card (middle) for analysis. The p-BNC card houses a miniaturized microbeadimmunoanalyzer with on-card samplemetering, reagent storage, and biohazardwaste disposal. Following sample introduction, the p-BNCcard is loaded intothe battery-powered analyzer (right) possessing mechanical, optical, and electronic components to drive the single analyte or MMP immunoassays tocompletion, followed by automated imaging, analysis, and readouts for access by the clinician.

Figure 2. The research-grade [A (I)]and the completely integratedp-BNCs [A (III)] retain identicalmicrobead immunoanalyzers housedin a bead holder [A (II)] permittingoptimization of individualcomponents and translation betweenthe systems. The p-BNC houses themicrochip [A (II)] and is constructedwith alternate layers of precision cutdouble-sided adhesive and laminatesto generate microfluidic features[A (I and III)]. Reagents and sample areuniformly delivered to the agarosemicrobeads [B (II)] and sandwichimmunoassays are completed in theagarose nano-nets [B (III)].Immunoschematic illustration[C (I–IV)] depicts sequential molecularevents on the agarose microbeadimmobilized with capturing antibody(cmAb) specific to the analyte ofinterest [C (I)], introduction of samplecontaining analyte (Ag) of interestfollowed by binding to the cmAb[C (II)], formation of a completedimmunomolecular sandwich [C (III)]with analyte-specific Alexa Fluor 488–coupled detecting antibody (dmAb),and signal visualization withfluorophore excitation [C (IV)], wherethe generated signal is proportional tothe analyte concentration. Unboundanalyte and detecting antibody areremovedwithhigh-stringencywashesfollowing steps II and III (not depicted).

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detection and output. These significant advances when fullydeveloped and placed intowidespread clinical practice havethe potential to permit untethering from laboratory sup-plies such as pipettes, reagent and buffer cold-chain, micro-scopes, and pumps, limitations associated with the tradi-tional laboratory infrastructure (Fig. 2A, I) to realize thePOC potential of the p-BNCs.

Here, we focus on the adaptation of the immunoanalyzermodule of the p-BNCs for rapid, sensitive, and precisequantification of serum CA125, as the proof-of-conceptbiomarker for ovarian cancer, with prominent implicationsin the multimodal and multimarker screening strategies, toderive the technical and operational specifications for theclinical-grade p-BNCs.

Optimization of p-BNC operating variablesLike ELISA, the p-BNC system uses sandwich immunoas-

says (Fig. 2C); however, immunocomplexes are formedthroughout the 3D bead matrix (Fig. 2B, III), rather thanon a 2D flat surface in ELISA. Here, the p-BNC–enhanceddimensionality in a dynamic (i.e., nonequilibrium) micro-fluidic environment yields new challenges related to assaydevelopment and optimization. Efficient biomarker cap-ture translating into high-performance immunoassays isinfluenced by a complex interplay of operating variables:epitope-guided matched pair screening, matched pair ori-entation, capturing and detecting antibody concentrations,sample and detecting antibody incubation times, and theflow rates corresponding to these incubation steps. Hence,these variables were individually assessed to derive optimalmatched pairs and operating conditions (technical specifi-cations) based on analytical performance criteria (slope,LOD, precision, and linearity).

Matched antibody pair selectionThe extracellular domain of CA125 shed in serum has

multiple 156 amino acid repeat domains, each containingthe 3 major nonoverlapping epitopes recognized by theOC125-like, M11-like, and OV197 antibodies (10, 39). Onthe basis of this established epitope mapping data, an arrayof antibodies were chosen to encompass the 3 distinctdomains on the molecule; OC125-like antibodies (clonesM002201 and M8072320), M11-like antibodies (clonesM77161, M80272321, and M002203), and OV197-likeantibody (clone M8072322). The matched pair identifica-tion for the CA125 sandwich immunoassay was enabled byscreening each of the aforementioned clones in both cap-turing and detecting formats, thus potentially testing 30such combinations.

"Nonrestrictive conditions" involving 30-minute incu-bation times for both the analyte and detecting anti-body to permit sufficient reaction times at slow flow rates(250 mL/min), a flow-through format to avail excessanalyte, relatively high analyte (100, 250, and 500 U/mL),and detecting antibody concentrations (1:250 dilution) toensure adequate signal visualization were used to enablematched pair identification under unoptimized conditions.A small subset of the results from this extensive study ispresented in Fig. 3A encompassing the matched pair select-ed eventually. Here, M8072320 (OC125) was chosen asthe detecting antibody and the results (MFI against CA125concentrations) represent the performance of the other5 clones as capture antibodies immobilized on the agarosemicrobeads (107 ng/bead concentration) to form a match-ed pair for CA125.

The performance gradient of the various antibody paircombinations was concordant with expectations based on

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Figure 3. [A] Calibration curves for a series of antibody pair combinations generated with clone M8072320 (OC125) as the detecting antibody and theclones shown in the legend as capturing antibodies. The optimal matched pair (clone M8072321 as the capturing antibody and M8072320 as thedetecting antibody) was chosen based on the highest slope exhibited by the corresponding calibration curve. The performance gradient of the variousantibodies was concordant with their associated epitope specificities. [B] Calibration curves showing the importance of matched pair orientation in aflow-based immunosensor system. Here, the correct orientation with clone M8072321 as the capturing moiety (and M8072320 as the detectingantibody) exhibited significantly higher performance in terms of slope and SNR in comparison with the reversed orientation where the same clone wasused for detection.

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epitope specificities. Antibodies binding to the samedomain and from the same clone performed the poorest(M8072320 capture and detection), represented by a rela-tively flat response in Fig. 3A and served as the negativecontrol with the lowest slope and signal-to-noise ratio(SNR) at lowest tested concentration. The antibodies rec-ognizing the same epitope, but from different clones(M002201 capture and M8072320 detection), exhibitedpoor, albeit slightly improved response above negativecontrol, suggestive of strong competition between theclones for the same epitope. The other matched pairsformed between the detecting antibody [M8072320(OC125)] with other capture antibodies recognizing dis-tinctly different domains (M11 and OV197) showed goodresponse over the tested range, with the performance gra-dient based on relative antibody affinities. M8072320(OC125) clone for detection paired with M8072321(M11) clone as the capture antibody exhibited the bestperformance in termsof aforementionedperformancepara-meters with the highest slope, SNR, and linearity among the30 tested combinations.The assignment of the capturing and detecting antibody

orientation for a given matched pair is critical to the p-BNCperformance as illustrated by Fig. 3B. Here, a matched pairformed with the optimal "correct orientation" performedsignificantly better than the "reverse orientation," where

the capturing and detecting moieties were interchanged, incontrast to results reported in the literature for flow cyto-metric bead–based immunoassays and ELISAs for identicalclones (40). Furthermore, microfluidic immunosensors,such as the p-BNCs, operate under dynamic (i.e., nonequi-librium) conditions, in contrast to the aforementionedsystems, necessitating extensive screening to arrive at opti-mal matched pairs and orientation as elucidated by thisstudy (40, 41).

Concomitant optimization of immunoreagentconcentrations

To avoid prebiasing based on order of optimization, thecapturing and detecting reagent concentrations were opti-mized concomitantly. "Nonrestrictive" incubation timesand flow rates as previously described were used. Agarosemicrobeads with 320, 107, and 5 ng/bead immobilizedcapturing antibody concentrations were multiplexed viaspatial recognition along each column (3 � 4 array), per-mitting simultaneous exploration of multiple reagent con-centrations and calibration curves were generated withvarious detecting antibody dilutions (1:250, 1:500, and1:1,000). Representative photomicrograph from this studyat 50 U/mL CA125, 1:250 detecting antibody dilution, andvarious tested capturing antibody concentrations are pre-sented in Fig. 4A.

Figure 4. [A] Typicalphotomicrograph showingadvantages of the p-BNCimmunosensor to simultaneouslyassess multiple capturing antibodyconcentrations in one experimentalrun. [B] A 3D surface plot showinginfluence of capturing and detectingantibodyconcentrations on the slopeof the resultant immunoassay. Thehighest slope is seen for thecapturing and detecting antibodyconcentration combinationcorresponding to the peak denotedby the orange zone (see color map).[C] Calibration curves showing theeffect of capturing antibodyconcentration on the slope of theCA125 immunoassay with higherconcentrations showing increasedslope at the detecting antibodydilution, exhibiting the highestlinearity. (�ve ctrl stands for negativecontrol.)

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A 3D surface plot was generated to simultaneouslyassess the effects of capturing and signaling antibodyconcentrations on the slope of the calibration curve(Fig. 4B). The slope was the highest with a capturingantibody concentration of 320 ng/bead at 1:250 detectingantibody dilution, but at the cost of linearity (R2 ¼0.7345). The effect of capturing antibody concentrationon the slope at the optimal detecting antibody concentra-tion (exhibiting the highest linearity) is depicted inFig. 4C. The upper and lower bounds for exploring detect-ing antibody dilutions were set on the basis of deviationfrom linearity at the upper end and unacceptably low SNRat the lower end. Similarly, concentrations lower than5 ng/bead of capturing antibody provided unacceptablylow SNR and concentrations higher than 320 ng/bead didnot offer any additional analytical advantages. This behav-ior is indicative of either the loading capacity of the beadsor lack of diffusion into porous beads and access to theadditional binding sites (generated with increased captureantibody concentration) due to steric hindrance resultingfrom the size of CA125. The optimal capturing and detect-ing antibody concentration combination with the highestlinearity (R2 ¼ 0.999) along with the maximum SNR andslope were chosen as 320 ng/bead and 1:500 dilution,respectively, for further experimentation.

Influence of flow rates and incubation timesThe flow rate/time dependence of reagent and sample

delivery was explored to identify whether these crucialdeterminants can be tuned to practical time constraintswithout loss of performance. As such, upper limit for thetotal assay time for the optimization steps describedpreviously was set at a maximum of 1 hour. Detectingantibody reagents have lower volume restrictions thanserum samples and hence the corresponding incubationtime was chosen first for optimization. With previouslychosen optimal matched pairs and reagent concentra-tions, incubation times varied from 10 to 30 minutesunder equimolar conditions (to preclude bias from lowerreagent availability). Longer incubation times resulted in

poor assay performance in terms of low slope and SNRalong with high imprecision (Fig. 5A). Given that thedetection step follows analyte capture by the immobilizedcapturing antibody (Fig. 2C, III), shorter incubationtimes are sufficient to complete the immunosandwich asindicated by these data. Longer incubation times andslower flow rates correspond to excess detecting antibodyin the pores and necessitate higher wash times to discardthe unbound reagent. However, wash times and washflow rates were held constant in this study leading to thepronounced effect of longer incubation times on theslope and precision. Furthermore, given the molecularsize of CA125, analyte capture is realized mainly on theperiphery of the bead (Fig. 4A), enabling the detectingreagent easy access and fostering shorter incubationtimes. Consequently, a detecting antibody incubationtime of 10 minutes was chosen as optimal as improve-ments in slope and precision resulting from loweringincubation times levels off at this time.

The sample incubation was carried out under the closedloop "recirculation mode" to increase analyte exploitation.A flow rate range of 250 to 1,500 mL/min was examined,translating into 7.5 to 45 passes (repeated introductionof the sample) to the array and the corresponding effecton slope is documented in Fig. 5B. The slope increasessteeply with increase in flow rate reaching a maximum at750 mL/min and then levels off. Initial increase in the slopecan be attributed to overcoming transport limitations andconsequently improved analyte exploitation, a frequentlyencountered situation inmicrofluidic systems (42). Beyond750 mL/min, the reaction-limited regimen prevails andhence improvements in slope level off at this time (43).The optimal flow rate was selected as 750 mL/min. Simi-lar trends were observed for lower immobilized con-centrations, albeit at different optimal "cutoff" flow rates(Fig. 5B). The corresponding incubation time was held at30 minutes as any further decrease resulted in unaccept-ably low SNR (data not shown). The CA125 p-BNC immu-noassay conducted with the optimal parameters was com-pleted in a total of 43 minutes inclusive of wash times

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Figure 5. [A] Effect of detectingantibody incubation times on theslope and SNR of the CA125p-BNCs. Decrease in incubationtime resulted in improvedimmunoassay slope along withcorresponding increase inprecision shown by the error bars.[B] Effect of sample incubation flowrates on the slope of the CA 125immunoassay. Initial increase inflow rate resulted in a steepincrease in slope through750 mL/min (optimal flow rate) forthe optimal capturing antibodyconcentration (320 ng/bead) andthen leveled off. Similar trendswerealso noted for other capturingantibody concentrations.

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making it ideally suited for implementation in a POCsetting.The LOD following each optimization step along with

the corresponding linearity and percent of coefficientof variance (%CV) is tabulated (Table 1). These resultsindicate performance improvement in terms of LODwith each step, with the most increase noted for thesample incubation flow rate optimization step. This isexpected as this step leads to increased analyte exploita-tion and is consistent with previous studies (44). Immu-noassay variables interact in a complex manner with each

other and the iterative approach presented here cannotaccount for these interactions and is subject to biasfrom the order of optimization steps. A statistical designof experiments method is currently being explored toaddress this issue.

Analytical validationUsing the optimized CA125 immunoassay, standard

curves were obtained over the range of 10 to 400 U/mL,pertinent to early disease detection. The calibration curvewas linear (R2 ¼ 0.99) over this range and the correspond-ing dose–response curve fitted to a 4-parameter logisticequation is shown in Fig. 6A.

The zero analyte concentrationwas evaluated in triplicateand the concentration corresponding to 3 SDs abovethe mean signal at zero was determined as the LOD. TheLOD of 1.0 U/mL was comparable with the values reported(0.05–1.45 U/mL range) for the currently available com-mercial systems (45).

To evaluate precision, serum-based tumor marker con-trol standards corresponding to low (21.1 U/mL), medi-um (64.2 U/mL), and high (186 U/mL) concentrationswere used. The intra-assay precision of the p-BNCs, denot-ing the variation between the analyte-sensitized beadsplaced within a chip (6 of 9 beads considered), wasevaluated for these 3 standards. The interassay precisionbetween the chips was estimated for the 3 standards over3 consecutive days. For the low, medium, and high stan-dards, the intra-assay precision (%CV) values were 1.4%,3.0%, and 1.3%, respectively, and the interassay precision(%CV) values were 1.2%, 1.5%, and 0.82%, respectively.The precision values obtained on the minisensor ensemblewere competitive with current commercial laboratory-based standards (45). The slightly higher intra-assayprecision is reflective of bead sensor homogeneity anduniform fluid delivery. Furthermore, the automatedp-BNC minimizes assay variations resulting in excellentinterassay precision. Finally, potential interference fromserum components were ruled out because of dilutionlinearity (R2 ¼ 0.98) tested for a preselected serum samplewith a high CA125 value (data not shown).

Table 1. Effect of each optimization step on theLOD and the corresponding linearity andprecision

Optimizationstep

LOD,U/mL

Linearity Avg%CV

Matched pairidentification

35.1 0.9982 5.04

#Detecting mAbconcentration

4.75 0.9999 3.69

#Capturing mAbconcentration

4.33 0.9952 2.92

#Detecting mAbincubation time

2.71 0.9930 4.26

#Sample incubation

flow rate0.24 0.9980 2.09

#Optimized assay 0.24 0.9980 2.09

NOTE: Results indicate improvement in LOD and precisionfor each optimization step with themost improvement notedfor the sample incubation flow rate step.

Figure 6. [A] Dose-response curvefor CA125 quantification on thep-BNCs over 10 to 400 U/mLconcentration range. [B] Plotshowing good correlation betweenthe p-BNCs and FDA-approvedELISA for values of CA125measuredin the sera of patientswith advanced-stage epithelial ovarian cancer.

250

200

150

100

50

01 10 100 1,000 1,000 2,000 3,000 4,000

CA125 ELISA (U/mL)Log CA125 (U/mL)

R2 = 0.97

MF

I

CA

125

BN

C (

U/m

L)

4,000

3,000

2,000

1,000

0

A B






Method validationTo show clinical use of the p-BNC, CA125 levels were

assessed in sera obtained frompatients with advanced-stageovarian cancer (n¼ 20). Sera (100 mL) were diluted 10-foldwith 0.1% PBSA and CA125 values were determined fromstandard calibration curves on the p-BNCs. Accounting fordifferences in calibrators between the methods, the resultsfrom this study are presented in Fig. 6B. The concentrationsobtained on the p-BNC correlated well with the currentFDA-approved gold-standard ELISA (R2 ¼ 0.97). Sampleswith CA125 more than 4,000 U/mL were analyzed, butexcluded from the results presented here because of thelimited number of data points in the region.

DiscussionThe p-BNC synergizes components from microfluidics,

biomarker discovery, clinical chemistry, and image analysis,and in this work, we have adapted the miniaturized inte-grated immunoanalyzer for specific applications in ovariancancer diagnostics. The p-BNC harnesses inherent micro-fluidic advantages of reduced assay times, reagent andsample economy upon multiplexing and inexpensive con-struction, alongwithmultiplexability andPOCamenabilitycompliant with requirements for novel early detection andscreening platforms for ovarian cancer.

We chose CA125 as the proof-of-concept biomarkerdue to its prominent implications in the MMS and MMPstrategies. For optimal matched pair identification, weinvestigated 30 antibody pair combinations and qualifiedthem on the basis of rigorous analytical response criteriafor the highest immunoassay slope, linearity, and preci-sion along with the lowest LOD (Fig. 3A and B). Wedelineated and optimized the variables affecting p-BNCperformance; reagent concentrations (Fig. 4B and C),sample and reagent incubation times (Fig. 5A), and thecorresponding flow rates (Fig. 5B). We tapped the multi-plexing potential of the p-BNC to simultaneously analyzemultiple antibody clones and reagent concentrations ona single microchip (Fig. 4A). Each optimization stepresulted in qualified improvement based on analyticalresponse criteria (Table 1). The LOD (1.0 U/mL) and theinter- and intra-assay precision (1.2% and 1.9%, respec-tively) values obtained were at least comparable or betterthan most currently available commercial systems (45).The optimal conditions here defined through this studywill now form the technical specifications for the "clinical-grade" p-BNCs.

The "clinical-grade" p-BNC retains identical microfluidicelements andmost importantly, the programmable agarosemicrobead core, permitting rapid and rigorous optimiza-tion to be translated for widespread distribution and testing(Fig. 2A). This credit card–sized p-BNC can interface withand has coevolved along with a compact (13.5 lbs) toastersize analyzer (Fig. 1), developed by our commercial part-ners, which uses light-emitting diode–based optics alongwith a miniaturized charge-coupled device (CCD) and amechanical actuator that circumvent the need for a micro-

scope and external pumps, as necessitated for a POC setup(36). In the completely integrated p-BNCs (Fig. 2A, III), theloaded sample is accurately metered via a sample loop,following which the card is inserted into the battery-pow-ered analyzer. Here, buffer containing blister packs arecrushed and sample and reagents are released by actuationof a stepper motor and delivered to the nano-porousagarose beads housed in a stainless steel chip for assaysequence completion. Biohazardous wastes are containedin the waste chambers housed on the p-BNCs. Upon assaycompletion, optical signal capture followed by automatedimage analysis results in an output displayed on the built-inscreen (37). Lack of such codeveloped scaled instrumen-tation has been a limiting factor for similar high-perfor-mance microfluidic systems to be distributed in the clinicalsettings and adapted to the POC (46), and this potentialto untether from expensive traditional laboratory infra-structure is a particularly distinguishing feature of thep-BNCs.

The high-quality analytical performance here reportedfor the p-BNC is achieved within 43 minutes, well suitedfor POC adaptation, permitted by the high surface areaand capture efficiency of the 3D agarose nanofibers com-pared with the traditional 2D platforms (38). Also, meth-ods such as the p-BNCs circumvent the need for central-ized laboratory infrastructure necessitated by ELISA,where turnaround times on the order of 24 to 48 hoursare typical with 3 degrees of separation between thepatient and the results (37). Traditional systems are fur-ther limited by one-marker-at-a-time approach and infea-sibility for POC adaptation. Further reductions in assaytimes and sample volumes are anticipated with higheranalyte capture efficiency, permitted by optimal beadholder chip geometry design powered by computationalfluid dynamics.

Method validation of the p-BNCs using sera of patientswith advanced-stage ovarian cancer showed good correla-tion (R2 ¼ 0.97) with the current gold-standard ELISAsystems (Fig. 6B). To show proof of concept, serum (asused in current systems) was chosen. To minimize sampleprocessing and to facilitate POC analysis, we are nowinvestigating the feasibility of whole-blood samplesobtained from a finger-stick to be applied directly to thep-BNCs via capillary loading. Future work will encompassdiseased samples at early and preclinical stages with corre-sponding age-matched negative controls to encompass thelow biomarker concentration range.

This work serves to define from a methods developmentperspective, the initial implementation of a chip-basedensemble for ovarian cancer biomarker testing at the POC.Further work is required to secure regulatory approval, anecessary and critical step before widespread clinical dis-tribution is possible. Thus, given the preliminary nature ofthiswork, it would be premature to claim full clinical use forearly disease detection at this stage. However, the CA125p-BNCs could envision use in a variety of scenarios. In thiscontext, CA125 continues to be the single-best biomarkerfor ovarian cancer despite the search for novelmarkers (47).

Raamanathan et al.





High analytical precision permitted by the CA125 p-BNCswill enable interpretation of biologic variations uncon-founded by analytical variations, necessary for MMS. Poorassay precision translates into poor biomarker perform-ance and these results are important in the light of highdegree of variation in biomarker assay precision (2%–58%)noted recently (47). In addition, CA125 results obtainedin 43 minutes can essentially permit TVS (if required) onthe same day preempting follow-up visits. Harnessing themultiplexing ability of the p-BNCs, consistent with theMMP approach, we are currently placing newly discoveredmarkers on to the p-BNCs alongside CA125 for increasedPPV (31). Such a system could be used for longitudinalmonitoring of multiple markers as a potential screeningmodality. Finally, the CA125 p-BNCs could potentiallyreduce the residence time in the "diagnostic pipeline par-adigm by validating novel biomarkers on the system alongwith serving as the endpoint diagnostic (23).Taken together, the CA125 p-BNC shows promising use

for ovarian cancer diagnostics, with competitive analyticalperformance metrics, reduced assay time, potential formultiplexing and POC amenability, necessary for large-scale implementation of early detection and screeningmethodologies.

Disclosure of Potential Conflicts of InterestR.C. Bast, Jr, is a consultant/advisory board member of Vermillion,

Fujirebio Diagnostics, and receives royalties for CA125. J.T. McDevitt hasan ownership interest and commercial research grant from LabNow and isa Consultant/Advisory Board member for Force Diagnostics and LabNow.

No potential conflicts of interests were disclosed by other authors. Thecontent is solely the responsibility of the authors and does not necessarilyrepresent or reflect views of CPRIT, the NIH, or the United StatesGovernment.

Authors' ContributionsConception and design: A. Raamanathan, G.W. Simmons, N. Christodou-lides, P.N. Floriano, S.W. Redding, R.C. Bast Jr, J.T. McDevittDevelopment of methodology: A. Raamanathan, G.W. Simmons, N.Christodoulides, J.T. McDevittAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): A. Raamanathan, G.W. Simmons, W.B. Furmaga,S.W. ReddingAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): A. Raamanathan, P.N. Floriano, J.T.McDevittWriting, review, and/or revision of themanuscript: A. Raamanathan, N.Christodoulides, P.N. Floriano, K.H. Lu, R.C. Bast Jr, J.T. McDevittAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): W.B. FurmagaStudy supervision: N. Christodoulides, J.T. McDevitt

Grant SupportFunding for this work was provided the Cancer Prevention and Research

Institute of Texas (CPRIT) (RP101382) as well as by the NIH through theNational Institute of Dental and Craniofacial Research (U01 DE015017 andU01 DE017793). This work was also supported by funds from the NationalCancer Institute (NCI) Ovarian SPORE (P5O CA 83639) and philanthropicsupport from Golfers against Cancer, the Tracey Jo Wilson Foundation, andthe Mossy Foundation.

The costs of publication of this article were defrayed in part by thepayment of page charges. The article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 9, 2011; revised March 2, 2012; accepted March 26,2012; published OnlineFirst April 9, 2012.

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2012;5:706-716. Published OnlineFirst April 9, 2012.Cancer Prev Res Archana Raamanathan, Glennon W. Simmons, Nicolaos Christodoulides, et al. Point-of-CareQuantification: Toward Ovarian Cancer Diagnostics at the Programmable Bio-Nano-Chip Systems for Serum CA125

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