ISSN 1359-7345

Chemical Communications

www.rsc.org/chemcomm Volume 49 | Number 72 | 18 September 2013 | Pages 7863–7970

COMMUNICATIONJun-Jie Zhu, Hui Wang et al.Ultrasensitive dual-channel detection of matrix metalloproteinase-2 in human serum using gold-quantum dot core–satellite nanoprobes

This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 7881--7883 7881

Cite this: Chem. Commun.,2013,49, 7881

Ultrasensitive dual-channel detection of matrixmetalloproteinase-2 in human serum usinggold-quantum dot core–satellite nanoprobes†

Tingting Zheng,ab Rui Zhang,a Qingfeng Zhang,b Tingting Tan,c Kui Zhang,c

Jun-Jie Zhu*a and Hui Wang*b

We have developed a robust enzymatic peptide cleavage-based

assay for the ultrasensitive dual-channel detection of matrix metallo-

proteinase-2 (MMP-2) in human serum using gold-quantum dot

(Au-QD) core–satellite nanoprobes.

Matrix metalloproteinases (MMPs) constitute a family of zinc-dependent endopeptidases that degrade the extracellular matrixproteins.1 The upregulation of MMPs has been found to be closelyassociated with pathological progression of cancer, which makes theMMPs important biomarkers for early cancer diagnosis and targetsfor therapeutic drug development.2 MMP-2, also known as gelatinaseA, has been identified as one of the key MMPs that are capable ofdegrading type IV collagen.3 Over-expression of MMP-2, typicallyevidenced by elevated MMP-2 concentrations in blood, has beenobserved in a variety of malignant tumors and the expression levelsand activities of MMP-2 are often correlated with tumor aggressive-ness.4 Dysregulation of MMP-2 expression has also been observed inhematological malignancies, such as human leukemia and myelo-dysplastic syndromes.5 Therefore, the quantification of MMP-2expression levels in blood samples is of vital importance to theclinical diagnosis and therapy at the early stage of cancer.

The low MMP-2 concentration levels and high complexity of theclinical samples impose the imperativeness of developing sensitive,selective, and reproducible MMP-2 assays with minimal interferencesfrom the complex biological sample matrices. Currently, the mostwidely used methods for MMP-2 detection in clinical samples areimmunoassays, such as enzyme-linked immunosorbent assay(ELISA)6 and surface plasmon resonance (SPR) immunosensing.7

Although these immunoassays offer satisfactory sensitivity and

selectivity, they involve tedious separation/washing processes andrequire the utilization of costly antibody proteins. Gel electro-phoresis-based gelatin zymography offers a simple yet powerfulanalytical tool for assessing the expression levels and activation statusof MMP-2 in biological samples;8 however, it is suited for qualitativerather than quantitative analysis. A series of protease assays, whichrelied on MMP-2’s capability to selectively hydrolyze peptidesubstrates containing a PLGVR sequence, have been developed forMMP-2 detection with the detection limit typically on the scale of1–200 ng mL1.9 Since the peptide cleavage is accompanied by theend-to-end distance change of the substrate peptide, fluorescenceresonance energy transfer (FRET)10 and bioluminescence resonanceenergy transfer (BRET)11 have been employed to develop MMP-2assays with detection limits of sub-100 ng mL1. Although the FRETand BRET assays can be performed homogeneously without separat-ing coexisting substances, an inherent drawback of these fluores-cence-based approaches is the interference by background signals incomplex clinical samples. Using upconversion fluorophores as theenergy donors for FRET-based MMP-2 assays, the background inter-ference can be effectively suppressed, pushing the detection limitdown to B10 pg mL1 range.12

Here we report an ultrasensitive enzymatic peptide cleavage-basedassay for the detection of MMP-2 in human serum with a detectionlimit as low as sub-pg mL1. The utilization of a Au-QD core–satellitenanoprobe allowed for dual-channel detection of MMP-2 based onboth anodic stripping voltammetry (ASV) and fluorescence measure-ments. As illustrated in Fig. 1A, the hybrid nanoprobes were fabricatedthrough assembly of multiple CdSe0.5Te0.5 QDs surrounding each Aunanoparticle core using single-stranded DNA oligonucleotides as thelinkers. Monodisperse CdSe0.5Te0.5 QDs (2.0 0.2 nm in diameter)and Au nanoparticles (10.2 0.5 nm) were both fabricated followingpreviously published protocols.13 The core–satellite structures werecharacterized using high-resolution transmission electron microscopy(HRTEM) (Fig. S1 in ESI†). The QD satellites exhibit clear lattice fringescorresponding to the (111) and (220) planes of a cubic phaseCdSe0.5Te0.5 alloy. The Au nanoparticle core has multitwined struc-tures, exhibiting clear (111) lattice fringes of cubic phase Au withvarious orientations. As shown in Fig. S2 in ESI,† the core–satellitenanoprobes exhibit combined extinction spectroscopic features of the

a State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry

and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210093, China.

E-mail: jjzhu@nju.edu.cnb Department of Chemistry and Biochemistry, University of South Carolina,

631 Sumter Street, Columbia, South Carolina 29208, USA.

E-mail: wang344@mailbox.sc.educ Department of Medical Laboratory, The Affiliated Drum Tower Hospital of Nanjing

University Medical School, Nanjing, Jiangsu 210008, China

† Electronic supplementary information (ESI) available: Experimental details andadditional figures and tables. See DOI: 10.1039/c3cc44623a

Received 20th June 2013,Accepted 11th July 2013

DOI: 10.1039/c3cc44623a

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7882 Chem. Commun., 2013, 49, 7881--7883 This journal is c The Royal Society of Chemistry 2013

Au core, the DNA linkers, and the QDs. Upon the formation of the core–satellite structures, the fluorescence of the QDs was significantlyquenched due to the close proximity between the QDs and the Aucore, as shown in Fig. S3 in ESI.† The QD satellites were furtherfunctionalized with streptavidin for subsequent conjugation to thesubstrate peptide.

Fig. 1B schematically illustrates the major steps involved in theconstruction of the MMP-2 sensor. The sensor was assembled on aneasy-to-make sensor substrate composed of a Au thin film over apolydimethylsiloxane (PDMS) elastomer.14 As shown in Fig. S4 in ESI,†this PDMS-Au sensor substrate is highly flexible, miniaturizable, andcan be made into arrays for high-throughput point-of-care diagnosticsusing, for example, the commercial 96-well plate as the template. Aspecifically designed biotinylated substrate peptide (biotin-Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys) was immobilized on the Au film through thethiol group of the C-terminal cysteine residue. MMP-2 is capable ofspecifically cleaving the peptide bond between Gly and Val,12 resultingin the loss of the biotin label at the N-termini, which diffused awayfrom the sensor substrate. Streptavidin-functionalized Au-QD core–satellite nanoprobes were then conjugated to the uncleaved peptidemolecules through the streptavidin–biotin interaction. The cadmiccomponent of the QDs exhibited sharp and well-resolved strippingvoltammetric signals.15 By dissolving the conjugated QDs with HNO3,the number of uncleaved peptide molecules, which was related to theMMP-2 concentration, could be quantified using ASV analysis. Mean-while, the quantum yields of some weakly fluorescent metal-sensitivedyes, such as Fluo-4 and Rhod-5N, significantly increased uponspecific binding of Cd2+, giving rise to dramatically enhanced fluores-cence signals.16 Such fluorescence enhancements can be used toquantify the Cd2+ concentration. Recruitment of multiple QD satellites

surrounding each Au core allowed for signal amplification in both theelectrochemical and fluorescence measurements as a direct conse-quence of the multiple-QD labeling strategy.

Atomic force microscopy (AFM) provided a straightforward way tocharacterize the peptide cleavage process. As shown in Fig. 1C andFig. S5 in ESI,† the Au film formed over PDMS was relatively compactand smooth. After immobilizing the peptide on the film surface,protrusions with a height of B15 nm became visible. After incuba-tion with MMP-2, a significant decrease in the height of the protru-sions was clearly observed, indicating the cleavage of peptidemolecules. The peptide cleavage process was also monitored usingASV. A distinct stripping voltammetric peak for the cadmium oxida-tion was observed at around0.78 V (vs. SCE) and the peak intensitywas determined by the total number of QDs in the nanoprobesattached to the uncleaved peptide molecules. The electrochemicalsignal intensity decreased gradually as the MMP-2 incubation timeincreased until it reached a plateau at 2 h (Fig. S6 in ESI†), suggestingcompletion of the peptide cleavage. The peptide cleavage activity ofMMP-2 could be effectively inhibited by 1,10-phenanthroline (Fig. S7in ESI†), which was reported as a common inhibitor of MMPs.17

As shown in Fig. 2A, the electrochemical signals measured afterthe completion of peptide cleavage decreased in intensity as theconcentration of MMP-2 increased. A linear relationship was foundbetween the relative change in the peak current, A%, and theconcentration of MMP-2, CMMP-2, within the concentration rangefrom 1 to 500 pg mL1. A% was calculated by A% = (I0I)/I0 100%,where I and I0 represent the peak currents obtained in the presence

Fig. 1 Schemes of (A) nanoprobe fabrication and (B) sensor construction.(C) AFM images of PDMS-Au (i) and the biotin-PLGVR/PDMS-Au before (ii) andafter (iii) the peptide cleavage.

Fig. 2 Results of (A) ASV and (C) fluorescence measurements at differentconcentrations of MMP-2 (from a to i: 0, 1, 10, 50, 100, 200, 300, 400 and500 pg mL1). Insets: the plots of A% and AF% versus the concentration ofMMP-2. (B) A% and (D) AF% of the biosensor toward various inspected species.The concentrations of MMP-2 and MMP-3 were 0.5 and 5.0 ng mL1, respectively.The concentration of all the other interfering species was 0.1 mM.

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This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 7881--7883 7883

and in the absence of MMP-2, respectively. The linear regressionequation was A% = 4.47 + 0.13CMMP-2, with a linear regressioncoefficient of 0.998 (n = 8). The limit of detection was calculated tobe 0.63 pg mL1 at 3s. The ASV detection displayed very goodselectivity for MMP-2 as shown in Fig. 2B.

The fluorescence enhancement of Rhod-5N dye induced byCd2+ allowed us to quantify the MMP-2 concentrations basedon fluorescence measurements. The fluorescence intensity ofRhod-5N decreased upon increasing the concentration of MMP-2(Fig. 2C). A linear relationship was found between the relative changein fluorescence intensity, AF%, and CMMP-2 within the concentrationrange of 1–500 pg mL1. Here, AF% = (F0F)/F0 100%, whereF and F0 represent the fluorescence intensities of the system inthe presence and the absence of MMP-2, respectively. The linearregression equation was AF = 0.14CMMP-2 + 1.3, with a linearregression coefficient of 0.998 (n = 8). The limit of detection wasdetermined to be as low as 0.72 pg mL1, and the sensor hadexcellent specificity toward MMP-2 (Fig. 2D).

The as-constructed sensors were directly used to quantify MMP-2in clinical samples. We examined six human serum samples, amongwhich three were obtained from healthy human beings and the otherthree were obtained from leukemia patients. Taking into considera-tion the normal MMP-2 levels in human blood samples and the linearresponse range of our method, the samples were diluted 10-foldbefore analysis. In Fig. 3, we directly compare the results obtainedfrom ASV and fluorescence measurements using the current approachto the results obtained using a commercial ELISA kit. The standarddeviations were obtained from parallel measurements performed onthree sensors. It was apparent that the MMP-2 concentration levels inthe serum samples of leukemia patients were higher than those of thehealthy people. This dual-channel MMP-2 assay gave reliable resultsthat were very consistent with ELISA. As shown in Table S1 in ESI,† therelative deviations between ASV and ELISA were in the range of4.5 to2.5%, and the relative deviations between the fluorescence assay andELISA were in the range of4.4 to 3.2%.

In summary, we have developed a dual-channel MMP-2 assaybased on both electrochemical and fluorescence measurements bycombining the action of enzymatic peptide cleavage with a rationallydesigned Au-QD core–satellite nanoprobe. Using this dual-channelsensing strategy, we have been able to detect MMP-2 in the concen-tration range of 1–500 pg mL1 with a detection limit down to

sub-pg mL1 (B10 fM), which is the lowest detection limit forMMP-2 quantification ever reported so far (see Table S2 in ESI†). Theultrahigh sensitivity allows one to remarkably reduce the consump-tion of clinical samples (B1 mL of serum samples required for eachmeasurement in the current case). This MMP-2 assay is of low cost andis easy to use, holding great promise as a new point-of-care diagnostictool for early detection of cancer and other diseases. By selecting thesubstrate peptide sequences, this approach can be readily developedinto a general method with high sensitivity, selectivity, and accuracyfor the detection of MMPs and other proteases in clinical samples.

T. Z. thanks the Chinese Scholarship Council for OverseasVisiting Student Fellowship. J.-J. Z. acknowledges the supportby National Basic Research Program of China (2011CB933502)and the NSFC (21020102038, 21121091). H. W. acknowledgesthe support by U.S. NSF CAREER Award (DMR-1253231) and theUniversity of South Carolina Start-up Funds.

Notes and references1 H. Nagase and J. F. Woessner, J. Biol. Chem., 1999, 274, 21491–21494.2 K. Kessenbrock, V. Plaks and Z. Werb, Cell, 2010, 141, 52–67;

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4 T. Turpeenniemi-Hujanen, Biochimie, 2005, 87, 287–297; B. Bauvois,Biochim. Biophys. Acta, Rev. Cancer, 2012, 1825, 29–36.

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10 S. Lee, E. J. Cha, K. Park, S. Y. Lee, J. K. Hong, I. C. Sun, S. Y. Kim,K. Choi, I. C. Kwon, K. Kim and C. H. Ahn, Angew. Chem., Int. Ed.,2008, 47, 2804–2807; J. H. Kim and B. H. Chung, Small, 2010, 6,126–131; D. Feng, Y. Y. Zhang, T. T. Feng, W. Shi, X. H. Li andH. M. Ma, Chem. Commun., 2011, 47, 10680–10682; J. Li, C. H. Lu,Q. H. Yao, X. L. Zhang, J. J. Liu, H. H. Yang and G. N. Chen, Biosens.Bioelectron., 2011, 26, 3894–3899; X. Wang, Y. Q. Xia, Y. Y. Liu, W. X. Qi,Q. Q. Sun, Q. Zhao and B. Tang, Chem.–Eur. J., 2012, 18, 7189–7195.

11 H. Q. Yao, Y. Zhang, F. Xiao, Z. Xia and J. H. Rao, Angew. Chem., Int.Ed., 2007, 46, 4346–4349; Z. Y. Xia, Y. Xing, M. K. So, A. L. Koh,R. Sinclair and J. H. Rao, Anal. Chem., 2008, 80, 8649–8655.

12 Y. H. Wang, P. Shen, C. Y. Li, Y. Y. Wang and Z. H. Liu, Anal. Chem.,2012, 84, 1466–1473.

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17 J. H. Uhm, N. P. Dooley, J. G. Villemure and V. W. Yong, Clin. Exp.Metastasis, 1996, 14, 421–433.

Fig. 3 MMP-2 concentration levels in human serum samples quantified usingASV, fluorescence, and ELISA measurements.

ChemComm Communication

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Electronic Supplementary Information for

Ultrasensitive dual-channel detection of matrix metalloproteinase-2 in human serum using gold-quantum dots core-satellite nanoprobes

Tingting Zheng,a,b Rui Zhang,a Qingfeng Zhang,b Tingting Tan,c Kui Zhang,c Jun-Jie Zhu,*,a

and Hui Wang*,b

a State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical

Engineering, Nanjing University, Nanjing, Jiangsu 210093, China b Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street,

Columbia, South Carolina 29208, United States c Department of Medical Laboratory, The Affiliated Drum Tower Hospital of Nanjing University

Medical School, Nanjing, Jiangsu 210008, China

* Corresponding authors. Emails: jjzhu@nju.edu.cn (J.-J. Zhu); wang344@mailbox.sc.edu (H. Wang)

Electronic Supplementary Material (ESI) for Chemical CommunicationsThis journal is © The Royal Society of Chemistry 2013

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S1. Experimental Details:

Materials and Reagents. PDMS monomer and curing agents were purchased from Dow Corning

(Midland, MI). 6-Mercapto-1-hexanol (MCH), 1-(3-(dimethylamino)-propyl)-3-ethylcarbodiimide

hydrochloride (EDC), N-hydroxysulfosuccinimide (NHS), and tris(2-carboxyethyl) phosphine

hydrochloride) (TCEP), 1,10-phenanthroline, bovine serum albumin, Glucose, IgG, Glutathione, and IL-

6 were purchased from Sigma-Aldrich (St Louis, MO). Chloroauric acid, trisodium citrate, imidazole,

KCl, and MgCl2 were obtained from Shanghai Chemical Reagent Co. (Shanghai, China). MMP-2 and

MMP-3 was obtained from Sino Biological Inc. (Beijing, China). The biotinylated PLGVR-peptide

(biotin-Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys) was synthesized and purified by Shanghai GL Biochem,

Ltd. (purity 95.98 %, molecular weight 984.22, Shanghai, China). The thiol- and amine-functionalized

DNA oligonucleotides with the sequence of 5’-SH-(CH2)6-ATG GAG ATG CTC ATC-(CH2)6-NH2-3’

were synthesized and purified by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). The

human serum samples were provided by Nanjing Drum Tower Hospital. TCNB buffer (50 mM Tris

with 10 mM CaCl2, 150 mM NaCl, and 0.05% Brij 35; pH 7.5) was used in the experiments. Ultrapure

water (18.2 kΩ resistivity, Milli-Q, Millipore) was used for all the experiments.

Apparatus. UV-vis extinction and fluorescence spectroscopic measurements were performed on a

UV-3600 spectrophotometer and a RF-5301PC fluorometer (Shimadzu, Kyoto, Japan), respectively.

High-resolution transmission electron microscopy (HRTEM) images were taken using a JEOL 2010

electron microscope at an accelerating voltage of 200 kV. Atomic force microscopy (AFM) images

were obtained on a SPI3800 controller operated in tapping mode with an acquisition frequency of 1.5

Hz and line density of 512.2 × 2 μm scans. Electrochemical measurements were performed on a CHI

660C workstation (Shanghai Chenhua Apparatus Corporation, China) with a conventional three-

electrode system composed of a platinum wire as the auxiliary, a saturated calomel electrode (SCE) as

the reference, and a glassy carbon electrode (GCE) as the working electrode.

Fabrication of Au-QDs Core-Satellite Nanoprobes. Water-soluble CdSe0.5Te0.5 alloy QDs with

average diameter of ~ 2 nm were prepared following a previously reported protocol.1 Colloidal Au

nanoparticles (NPs) with average diameter of ~ 10 nm were prepared as reported previously.2 The

nanoprobes were fabricated by assembling multiple QDs surrounding each Au nanoparticle using

single-stranded DNA as the linker between the Au core and the QDs. First, 100 μL of 0.1 M imidazole

solution (pH 6.8) was added to 3’-amine- and 5’-thiol-capped DNA oligonucleotides (1 O.D.) for 30

min, followed by the addition of 40 μL of 0.1 M EDC and 2 mL of colloidal QDs. The mixture was

incubated at room temperature for 12 h with magnetic stir. The DNA-conjugated QDs were then

isolated from the reaction mixture and washed with H2O three times by centrifugation at 6000 rpm for

10 min at 4°C, followed by activation with 10 mM tris-(2-carboxyethyl) phosphine hydrochloride

(TCEP) for 30 min. After the pH of the Au colloids was adjusted to 9.2 using 0.1 M NaOH, freshly

activated DNA-conjugated QDs were added and the mixture was kept under gentle shaking for 30 min

at 10°C. The mixture was centrifuged at 15000 rpm for 30 min to remove the excessive DNA-QDs

conjugate particles that were not attached to the Au NPs. Finally, 1.0 mL of Au-DNA-QDs particles

were mixed with 3.2 mg of EDC and 0.1 mg of streptavidin in 50 mM of MES buffer (pH 5.2),

incubated for 2 h at room temperature under shaking, and kept undisturbed overnight at 4°C. The

reaction mixture was centrifuged at 15000 rpm for 5 min three times, and the supernatant was discarded.

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The obtained Au-QDs core-satellite nanoprobes were separated from the reaction mixture and washed

with phosphate buffered saline (PBS) through centrifugation at 15000 rpm for 5 min three times, and

finally redispersed in 1.0 mL of PBS (pH 7.4) and stored at 4°C.

Sensor Construction. PDMS monomer and the curing agent were first added to the 96-well plate

in a proportion of 1:0.06 and cured at 70°C for 2 h. HAuCl4 (1%) were dropped into each well and

incubated at 37°C for 48 h to form PDMS-Au composite film.3 All resulting wells were washed with

H2O three times, and stored at 4°C when not in use. The biotin-PLGVR peptide was activated with 1.5

μL of 10 mM TCEP in pH 5.2 acetate buffer for 1 h to prevent the terminal cysteine from forming

disulphide bonds. Afterwards, 50 μL of 1 mM biotin-PLGVR peptide was spread evenly over the

PDMS-Au composite film for 12 h at 4°C in 100% humidity, followed by immersion in 1 mM MCH for

1 h to remove the nonspecific adsorption. Subsequently, 100 μL of MMP-2 at various concentrations

was added to each well and incubated for 2 h at 37 °C. After washing with TCNB, 40 μL of diluted Au-

QDs core-satellite nanoprobes were dropped onto each well for 2 h to attach a nanoprobe onto each

uncleaved peptide molecule. Prior to electrochemical and fluorescence measurements, the biosensor was

washed with TCNB to remove all physically adsorbed nanoprobes.

Electrochemical and Fluorescence Measurements. After the binding of nanoprobes, 200 μL of

0.1 M HNO3 solution was dropped into each well for 2 h to completely dissolve the QDs in the captured

nanoprobes. For electrochemical analysis, the resulting solution was mixed with 1.0 mL of 0.2 M HAc-

NaAc buffer (pH 5.2) and 20 μL of 500 μg mL-1 Bi3+ solution to perform anodic stripping voltammetric

detection with a conventional three-electrode system composed of a platinum wire as auxiliary

electrode, a saturated calomel electrode (SCE) as the reference, and a glassy carbon electrode as the

working electrode. The anodic stripping voltammetric detection involved electrodeposition at -1.1 V for

120 s while the solution was stirred and stripping from -1.1 to -0.2 V under N2 atmosphere using a

square-wave voltammetry, with a frequency of 25 Hz, amplitude of 25 mV, and potential step of 4 mV.

For fluorescence analysis, the HNO3-treated solution were diluted with phosphate buffer solution

and centrifuged at 1000 rpm for 5 min to obtain a supernatant. After adjusting pH to 7.4 using 1 M

NaOH, the resulting solution containing 3 μM Rhod-5N was detected at room temperature with the

excitation wavelength of 540 nm.

ELISA for MMP-2 Detection. Concentrations of MMP-2 in serum samples were detected by

measuring absorbance changes at 450 nm using a commercial human MMP-2 ELISA Kit purchased

from Beijing Biosynthesis Biotechnology Co. Ltd. (Beijing, China). A standard curve for the

spectrophotometric procedure was obtained following the manufacturer’s protocol in the concentration

range from 0 to 12 ng mL-1. Briefly, the serum sample or MMP-2 standard solution (0-12 ng mL-1)

were added to the ELISA plate (50 μL/well) and incubated for 1 h at 37 ºC. Then each well of the plate

was washed five times with 200 μL of wash buffer. Enzyme-labeled bioconjunction solution was added

to each well and incubated for 60 min at 37 ºC and washed again with wash buffer. Subsequently,

substrate solution (50 μL solution A and 50 μL solution B) was added to each well and incubated for 15

min in the dark. Without washing the plate, 50 μL of stop solution was added to each well to stop the

color reaction. The absorbance was read immediately on an automated plate reader (model 680, Bio-

RAD) at 450 nm.

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S2. Figures:

Fig. S1 HRTEM image of a Au-QDs core-satellite nanoprobe. The QDs are indicated by circles. The lattice spacings of the (111) and (220) planes of cubic phase CdSe0.5Te0.5 and the lattice spacing of the (111) plane of cubic phase Au are labeled.

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Fig. S2 UV-vis extinction spectra of DNA, QDs, QDs-DNA, Au NPs, and Au-DNA-QDs core-satellite particles. Inset: Picture of colloidal suspensions of Au NPs, Au-QDs core-satellite particles, and QDs under room light.

Fig. S3 Fluorescence spectra (λexcitation = 300 nm) of QDs, QDs-DNA, and Au-QDs core-satellite particles. Inset: Picture showing the emission from colloidal Au NPs, Au-QDs core-satellite particles, and QDs under UV light excitation.

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A B

C

Fig. S4 (A) Picture of the PDMS-Au composite films (sensor substrate) made in a 96-well plate. (B) Picture showing the relative size of a sensor substrate to that of a dime (Unite State coin). (C) Picture showing the flexibility of the sensor substrate.

PDMS-Au PDMS-Au-peptide PDMS-Au-peptide after cleavage

Fig. S5 AFM images of the PDMS-Au (left), biotin-PLGVR/PDMS-Au before (middle) and after (right) the peptide cleavage. The bottom panels show the height profiles along the green lines in the corresponding AFM images in the top panels.

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Fig. S6 Time evolution of the ASV currents during peptide cleavage in the presence of 0.5 ng mL-1 MMP-2.

Fig. S7 ASV curves obtained after incubating the biotin-PLGVR/PDMS-Au with (a) 0.5 ng mL-1 MMP-2, (b) 0.5 ng mL-1 MMP-2 + 10 μM 1,10-phenanthroline, (c) buffer, and (d) 10 μM 1,10-phenanthroline for 2h.

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S3. Tables:

Table S1 Assay results of the clinical human serum samples.

Serum Samples

ELISA [a] (ng mL-1)

ASV [a] (ng mL-1)

Relative Deviation

(%)

Fluorescence Analysis [a] (ng mL-1)

Relative Deviation

(%)

S1 2.72 + 0.137 2.71 + 0.106 -0.369 2.65 + 0.107 -2.44

S2 2.02 + 0.164 2.06 + 0.0885 2.45 2.055 + 0.0720 1.98

S3 1.73 + 0.0661 1.75 + 0.221 1.34 1.76 + 0.0224 1.74

S4 4.54 + 0.261 4.51 + 0.174 -0.665 4.51 + 0.0725 -0.716

S5 4.23 + 0.271 4.14 + 0.0376 -2.00 4.36 + 0.128 3.15

S6 5.32 + 0.138 5.08 + 0.0466 -4.49 5.09 + 0.00893 -4.32

[a] Average values of three measurements.

Table S2 Detection limits of various methods for MMP-2 detection.

Detection method Detection limit Reference

Gel electrophoresis-based protease assays 1-6 ng mL-1 4

Protease assays using magnetic nanoprobes 69 ng mL-1 5

Magnetic resonance imaging techniques 170 ng mL-1 6

FRET between graphene oxide and QDs 70 ng mL-1 7

FRET between dyes and Au nanoparticles 12.5 ng mL-1 8

FRET between graphene oxide and fluorophores 3.5 ng mL-1 9

Bioluminescence resonance energy transfer (BRET) 2 ng mL-1 10

Upconversion FRET 10 pg mL-1 11

Dual-channel detection using Au-QDs core-satellite nanoprobes sub-pg mL-1 This work

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ESI-p9

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