Microfluidic multiplex biochip based on a point-of-care electrochemical detection system for matrix metalloproteinases

    Microfluidic multiplex biochip based on a point-of-care electrochemical detection system for matrix metalloproteinases Seung Yong Hwang, In Jae Seo, Seung Yong Lee, Yoomin Ahn PII: DOI: Reference:

S1572-6657(15)30073-4 doi: 10.1016/j.jelechem.2015.08.015 JEAC 2239

To appear in:

Journal of Electroanalytical Chemistry

Received date: Revised date: Accepted date:

10 April 2015 24 July 2015 10 August 2015

Please cite this article as: Seung Yong Hwang, In Jae Seo, Seung Yong Lee, Yoomin Ahn, Microfluidic multiplex biochip based on a point-of-care electrochemical detection system for matrix metalloproteinases, Journal of Electroanalytical Chemistry (2015), doi: 10.1016/j.jelechem.2015.08.015

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Microfluidic multiplex biochip based on a point-of-care electrochemical

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detection system for matrix metalloproteinases

Division of Molecular and Life Science, Hanyang University, and GenoCheck Co. Ltd.,

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Seung Yong Hwanga, In Jae Seob, Seung Yong Leea, Yoomin Ahnb

Ansan, Kyeonggi-do, Korea.

Dept. of Mechanical Engineering, Hanyang University, Ansan, Kyeonggi-do, Korea.

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Correspondence: Professor Yoomin Ahn, Department of Mechanical Engineering, Hanyang

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University, 55 Hanyangdaehak-ro, Sangnok-gu, Ansan, Kyeonggi-do, 426-791, Korea E-mail: [email protected],

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Fax: +82-31-436-8122

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Abstract

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Conventional cancer diagnostic techniques are not suitable for point-of-care testing because

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they are usually expensive, time consuming, labor intensive, and require large equipment. This paper presents a proof-of-concept biochip for point-of-care testing based on multiplex

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electrochemical detection. The microfluidic biochip is composed of inexpensive photoresistpolydimethylsiloxane layers over a glass substrate. The substrate has an Au working

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electrode and a Pt counter/reference electrode. A microchannel was fabricated in the photoresist layer and a micromixer was fabricated in the polydimethylsiloxane layer. Matrix

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metalloproteinases (MMP) 2 and MMP7 are used as biomarkers for ovarian and colorectal

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cancer diagnosis. An unlabeled peptide specific to MMPs is immobilized on an Au electrode

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and then impedance was measured using electrochemical impedance spectroscopy with electrolytes. As the MMP sample is injected into the biochip, the peptide is cleaved by enzyme hydrolysis and the impedance changes. MMP2 and MMP7 are quantitatively

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detected concurrently by measuring the variations of impedance. Using model samples, the detection ranges of the biochip were 0.1-400ng/mL and 0.001-100ng/mL for MMP2 and MMP7, respectively, and the overall assay time was reduced to about 1 h. The results indicate that a similar MMP-based electrochemical multiplex biochip could be used for the point-ofcare diagnosis of many forms of cancer.

Keywords: Cancer diagnosis, Electrochemical impedance spectroscopy, Enzyme hydrolyzation, labelfree peptide, PDMS-glass chip

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1 Introduction

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Early detection of cancer significantly improves patient outcomes [1]. Therefore, fast and

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simple low-cost diagnostic methods that are readily available and usable anywhere and at any

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time are required. One promising solution involves the development of cancer diagnostic point-of-care testing (POCT) biochips. Polymer-based microfluidic chips are the most

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suitable as POCT biochips, because they are low-cost, portable devices that require only a short check time and a small sample [2]. Cancer biomarkers are an essential factor for precise,

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quick, and low-cost detection of a particular cancer with POCT biochips. Biomarkers used in most existing cancer diagnosis are proteins, DNA methylation, miRNAs, ctRNAs, and

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circulating tumor cells [3]. Biosensors for protease assays have been reported because a

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thorough cancer diagnosis is possible if proteases, which are enzymes important for cancer

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initiation and metastasis, are used as biomarkers. Protease detection by conventional laboratory equipment uses immunohistochemistry [4], enzyme-linked immunesorbent assay (ELISA) [5], and gelatin zymography [6]. These methods are complicated, time consuming,

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and require expensive equipment. Some cancers require physical examination because most patients with early-stage disease do not display symptoms [7-9]. In particular, colorectal and ovarian cancers are often found in an advanced stage. MMP2 [10], cancer antigen 125 [11], and lysophosphatidic aid [12] are used as ovarian cancer biomarkers, while MMP7 [13], carcinoembryonic antigen [14], and syndecan [15] are used as biomarkers of colorectal cancer. The matrix metalloproteinase (MMP) family is comprised of zinc-dependent endopeptidases, a type of protease that hydrolyzes many extracellular matrix proteins; they are also involved in metabolism in vivo [16]. The MMP family is an important biomarker for the initiation, progression and transition of various cancers [10]. MMP2 and MMP7, in particular, may be effective biomarkers for

ACCEPTED MANUSCRIPT ovarian and colorectal cancers, respectively [13]. MMP2 destroys the basilar membrane in the initial stage of tumor infiltration of ovarian cancer, and may therefore be useful for early

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diagnosis. The presence of trace amounts of MMP7 may be able to diagnose colorectal cancer

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[17].

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Recent studies have examined biosensors that can detect MMPs. Sensing systems have been developed to detect MMP2 and MMP7, using bioluminescence resonance energy

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transfer between a quantum dot and bioluminescent proteins [18]. Biosensors have also been developed to sense proteases, such as MMP7, thrombin, and caspase-3, by fluorescence

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resonance energy transfer on a glass substrate [19]. MMP1 has been detected by a piezoelectric immunosensor [20]. These protease assay studies were for the purpose of

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precise basic research, but they are impractical for POCTs. Other assay techniques which are

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applicable to an MMP biomarker-based cancer diagnostic biochip include the electrochemical

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immunoassay (MMP9 [21] and MMP2 [22]) and surface plasmon resonance (MMP3 [23]). Immunoassays are highly accurate because they use an antigen-antibody reaction, but these tests are complicated and take a long time [24]. Examination by surface plasmon resonance

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can be performed inexpensively and quickly, but a costly optical detector is needed. A suitable method for the POCT biochip is the electrochemical measurement, which uses simple quantitative measurement while minimizing sample damage. In addition, miniaturization of the biodevice is easy because electronic circuits can be integrated with the biochip. MMP9 [25] and MMP2 [26] have been detected efficiently using proteolytic digestion of the peptide with an accurate three-electrode electrochemical method. However, these methods were optimized for the research lab, not POCT, and require time and effort for sample preparation and peptide modification. In a previous study, we reported a biosensor detecting MMP7 for the diagnosis of colorectal cancer [27]. Precise measurements, however, were limited because the

ACCEPTED MANUSCRIPT spectrometric procedure was used. We have since developed a simple 2-electrode electrochemical POCT biochip that can precisely detect MMP2 and MMP7 and is suitable for

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early diagnoses of ovarian and colorectal cancers. Furthermore, by measuring the quantity of

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MMP2 and MMP7 with a nonspecific hydrolysis (control), we sought to reduce the test time

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and increase the efficiency, so that various POCT applications are possible. We adopted hydrolysis action with the peptide, which has been reported as most effective way for MMP

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detection. The time needed to prepare biochips for detection was reduced by using unmodified peptide without any label. A relatively inexpensive glass-polydimethylsiloxane

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(PDMS)-based biochip was used to make a disposable microfluidic biosensor. A microfluidic mixer was integrated on the biosensor chip to enhance sensitivity by accelerating the reaction

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between sample and reagent. Immobilization of the peptide on the electrode surface and

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reaction conditions of the enzyme were optimized so that the right signal is sensed by the

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proposed biochip. Finally, our experimental performance evaluation showed that MMPs were successfully detected at the same time.

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2 Materials and methods

2.1 Detection principle

An electrochemical impedance spectroscopy (EIS) assay was used for electrochemical measurement. EIS assays are effective for detecting changes in charge transfer dynamics on the electrode surface using a bioactive layer placed on the electrode [18]. Reaction mechanisms inside the biochip that obtain the concentrations of MMP2 and MMP7 in diagnostic samples of ovarian and colorectal cancers through a change in impedance are shown in Fig. 1. First, partially digested peptides which respond specifically to MMP2 and

ACCEPTED MANUSCRIPT MMP7 are injected in the biochip. Different peptides are required for MMP2 and MMP7. The injected peptides are immobilized on the Au working electrode surface with a sulfur-gold

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reaction (Fig. 1(a)). Electrolyte is injected and impedance between the electrodes is measured

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(Fig. 1(b)). Impedance between two electrodes increases with the amount of peptide

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immobilized on the device because ionic movement from electrolyte into the electrode is obstructed. Then, a sample including MMPs is flown onto the peptide-modified electrode.

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MMP2 and MMP7 recognize the specific peptide synthesized and cleave it by hydrolysis (Fig. 1(c)). Then, the cleaved peptide is left on the working electrode, and is cleaved further

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depending on the amount of the target MMP in the sample. Electrolyte is injected again, and impedance between two electrodes is measured (Fig. 1(d)). Ionic movement is less obstructed

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by the cleaved peptides on the chips. Consequently, the impedance between two electrodes is

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reduced more than before cutting of the peptide. The concentration of the target biomarker in

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the sample is calculated from the relative impedance before and after peptide cleavage.

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2.2 Microdevice design

We invented a multi-detection microfluidics chip, which can electrochemically detect two biomarkers at once. In our previously developed biochip, the channel length was enlarged and the electrode was designed using an interdigitated finger configuration, such that two biomarkers were able to be detected [28]. However, sample flow was difficult to control because of the length of the channel and efficiency was not greatly improved compared to that of electrodes with a complicated and long shape. When channels of a microfluidic chip become longer, the reaction area increases, but sample flow becomes difficult to control. Hence, we kept the channel length constant but increased the width. The width and height of the designed channel were 2 and 0.1 mm, respectively. A two-electrode system with a simpler

ACCEPTED MANUSCRIPT structure than the three-electrode system was adopted for a disposable device. Chip size increases since electrodes become complicated in an interdigitated finger configuration to

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create a broad response area, if electrodes are placed along the width of the channel.

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Therefore, two electrodes were designed on the channel bottom parallel to the length of the

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channel to create an electrode surface area sufficient for the measuring reaction. The working electrode was 0.3 mm wide and 15 mm long. The width and length of the Pt

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reference/counter electrode were 0.6 and 15 mm, respectively. The distance between electrodes was 1 mm. A schematic drawing of the chip designed in this way is shown in Fig.

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2. To create a wider reaction area, agitation was added to the testing process in order to make the enzyme reaction more active. Accessibility of enzymes to peptides concentrated on the

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electrode can be improved by such agitation because in a micro scale channel, fluid does not

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flow turbulently, but rather laminarly. Thus, the micromixer helps to create better contact

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between MMPs and peptides. A chaotic passive micromixer of the micro-ridges pattern was created on the upper side of the channel to improve stirring efficiency [29]. The mixer height was 50 m so that sample flow within a 100 m high channel was not impeded. To increase

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reliability, a chamber (chamber-II in Fig. 2) for the control was added in addition to two chambers (chamber-I and chamber-III) for detection of target biomarkers.

2.3 Biochip fabrication

The biochip was fabricated based on an existing PDMS-glass hybrid microchip micromachining method [30, 31]. The glass substrate had Au and Pt electrodes on its surface, and a photoresist microchannel was also built over it. The manufacturing process is as follows. A positive photoresist (AZ5214, MicroChemicals GmbH, Germany) was spincoated on a glass wafer and patterned by photolithography. Platinum film was deposited over the

ACCEPTED MANUSCRIPT patterned photoresist by E-beam evaporation. Next, the photoresist was lifted off by acetone, forming a Pt electrode. A gold electrode was also produced the same way. Then, a negative

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photoresist (SU-8, MicroChem, US) was spincoated over the glass substrate formed with

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electrodes, and then the microchannel was formed by photolithograpy. In the PDMS layer, the

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gates and pectinate micromixer were machined as follows. The mold shape of the PDMS layer was patterned over a silicon wafer with an SU-8 photoresist. PDMS (Sylgard 184, Dow

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Corning, US) was poured over the SU-8 mold and hardened by curing at 60 ℃ for 4 hrs to

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produce the PDMS layer. After surface treatment with O2 plasma, glass substrate and PDMS layer were bonded and were heated for an hour in an oven at 100˚C. The biochip was

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completed, and the final fabricated chip is shown in Fig. 2(d).

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2.4 Preparation of peptide, enzyme, and electrolyte

The peptide sequence specific to MMP2 which was used in experiments is Gly(G)-Ser(S)-

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Arg(R)-Leu(L)-Ser(S)-Val(V)-Pro(P)-Ile(I)-Cysteamine, and the sequence of the peptide specific to MMP7 is Arg(R)-Pro(P)-Leu(L)-Ala(A)-Leu(L)-Trp(W)-Arg(R)-Ser(S)-Lys(K)Cysteamine [32]. The peptides were synthesized by Peptron Co., Ltd. (Korea). Amino acids were coupled one-to-one from the C-term using Fmoc solid-phase peptide synthesis. In order to couple cysteamine (HS-CH2CH2NH2) to the C-term of the peptide, synthesis was facilitated using NH2-Cysteamine-2-Cl-Trityl resin. Later, cysteamine was attached to the Au electrode via the thiol (HS) group, as it has been extensively reported [33]. The immobilization mechanism results in the bonding of the sulfur atom to gold and not the cleavage of the H-S bond. The bond is strong, since the closest packed monolayers of this absorbate grow epitaxially on the Au surface. MMP2 and MMP7 were purchased from

ACCEPTED MANUSCRIPT Calbiochem (Germany). In consideration of concentration ranges (details in Supplementary material Table S1) for normal and patient samples [34-38], MMP2 was prepared at

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concentrations of 0.0005, 0.05, 5, 50, 100, and 200 ng/mL and MMP7 was prepared at 0.0005,

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0.005, 0.05, 0.5, 5, and 50 ng/mL. These concentrations of MMP2 and MMP7 were mixed,

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such that six mixed enzyme solutions were made: 0.0005 ng/mL MMP2 and 0.0005 ng/mL MMP7, 0.05 ng/mL MMP2 and 0.005 ng/mL, 5 ng/mL MMP2 and 0.05 ng/mL MMP7, 50

MMP2 and 50 ng/mL MMP7. peptide

was

diluted

using

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mM

HEPES

(4-(2-hydroxyethyl)-1-

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The

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ng/mL MMP2 and 0.5 ng/mL MMP7, 100 ng/mL MMP2 and 5 ng/mL MMP7, 200 ng/mL

piperazineethanesulfonic acid), which was made by mixing 5 mM CaCl2 (Sigma-Aldrich) and

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150 mM NaCl (Sigma-Aldrich) in a ratio of 1:1; pH was controlled at 7.4 with HCl and

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NaOH. For enzyme dilution, 10 mM HEPES was used and the pH was controlled at 7.4.

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Washing was carried out using the peptide dilution buffer. The electrolyte solution for impedance measurement was made by mixing 160mM potassium chloride (Amersco), 80mM potassium ferrocyanide(III) (Sigma-Aldrich), and 80mM potassium hexacyanoferrate(II)

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(Sigma-Aldrich) in a ratio of 1:1:1.

2.5 Analytic procedures

Testing for MMP detection was performed with standard experimental conditions (see Supplementary material Table S2). First, electrolytes are put in the biochip through gate-II, so that all chambers are filled. Impedance is measured in the bare electrode. After that, the electrolyte in channels is removed through gate-II using a micro pipette, and peptides which respond specifically to MMP2 and MMP7 are injected into gate-I and gate-III, respectively, to immobilize them on electrodes in chamber-I and chamber-III. At this time, in order to keep

ACCEPTED MANUSCRIPT peptides from flowing into chamber-II, gate-III is closed when peptides specific to MMP2 are injected into chamber-I. The peptide solution is then injected through gate-I until chamber-I is

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filled. In a similar way, when peptides specific to MMP7 are immobilized, gate-I is closed

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and the peptide solution is injected through gate-III. After the immobilization, washing is

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done by pouring the buffer in all chambers through gate-II. Electrolyte is injected again through gate-II and impedance is measured in the peptide modified electrode. After signal

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measurement, electrolyte is eliminated through gate-I and gate-III by suction with a micropipette. Then, samples of MMP2 and MMP7 are put in all the chambers. After 15 min

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of the 30 min reaction time has elapsed, a micropipette is stuck in gate-II and the sample is agitated by resuspending five times. Lastly, electrolyte is poured through gate-II and

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impedance is measured after the enzyme reaction. The electrochemical assay was executed

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using a potentiostat (Ivium-n-Stat, Ivium Technologies, Netherlands).

3 Results and discussion

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3.1 Optimum conditions for electrochemical detection

A marked difference between impedance was not verified according to MMP concentration, when a preparatory experiment was performed at a peptide concentration of 10 g/mL. Thus, the 500 g/mL concentration was selected to clearly identify the difference between impedance. Detection conditions for the optimal electrochemical signal in multiplex biochips are immobilization time, washing time, and MMP enzyme reaction time. To find these optimum conditions, a 1:1 mixture of MMP2 (100 ng/mL) and MMP7 (100 ng/mL) was used as enzyme solution because cyclic voltammetry (CV) was most stable when MMP concentration was 100 ng/mL.

ACCEPTED MANUSCRIPT Peptide immobilization on the Au electrodes, was performed for 1, 5, 10, 15, 30, and 60 min, and then maximum currents were measured by the CV technique. The measured currents

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were compared with those of a bare electrode. The results shown in Fig. S1 (Supplementary

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material) confirm that the immobilization reaction happened reliably. When the

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immobilization time was 1, 5, 10, 15, 30, and 60 min, MMP2 showed 15.7, 26.7, 48.6, 32.9, 62.9, and 55.8 rate of change of the maximum current before and after immobilization.

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MMP7 showed a 32.6, 45.8, 27.6, 38.5, 24.8, and 38.6 % rate of change at these times (Table S3, Supplementary material). Though MMP2 and MMP7 had the biggest change rate at the

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30 and 5 min time points, respectively, this was unstable since standard deviations were relatively big. Because the dual detection has to be done concurrently on a chip, a 15 min

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immobilization time was used, because the standard deviations were reasonable and the rate

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of change of the current large for both MMPs. At this time, in order to exclude the influence

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of washing time, electrolyte was put in right after injection of the washing buffer and then signal was measured. Peptide was immobilized for 15 min, and the chambers of the biochip were washed for 1, 2, 5, 10, 30, and 60 min. Then, the maximum current was measured by

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the CV technique and compared with a bare electrode. The rate of change of the maximum current for MMP2 was 47.3, 40.7, 31.4, 48.6, 53.0, and 35.5 % at the washing times of 1, 2, 5, 10, 30, and 60 min, respectively (Table S4, Supplementary material).. The rate of change for MMP7 was 58.5, 35.7, 55.0, 30.7, 23.8, and 40.4 % at these time points. The washing time was selected as 1 min, where rate of change was stable and large for both MMPs on average. Peptide was reacted with MMP for 30 and 60 min after immobilization and washing under the determined optimum conditions, and maximum current was gauged by CV again. A comparison of the maximum current was made before and after the peptide-MMP reaction. The current changed to 240.5 and 96.0 % for MMP2, and 150.3 and 128.8 % for MMP7, at reaction times of 30 and 60 min, respectively (Table S5 Supplementary material). Reaction

ACCEPTED MANUSCRIPT time was decided as 30 min because the current variation was stable. A cyclic voltammogram measurement during a 15 min immobilization, 1 min washing, and 30 min enzyme reaction as

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determined by the optimum conditions is shown in Fig. 3. MMP2 shows big current

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measurements compared with MMP7. Measured results were taken with a bare electrode

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surface, after peptide modification, and after the peptide reaction with MMPs. The presence of a Faradaic process is not obvious in Fig. 3. A pair of redox peaks, however, is evidenced

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when the electrode surface is bare and the peptide is cleaved by MMPs, and a redox wave is not obtained after the surface is modified by peptides. A similar cyclic voltammogram was

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reported via trypsin assay using proteolytic digestion of the peptide [39]. The measured currents at each case are clearly distinguished from each other. Consequently MMP detection

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on the developed biochip is possible.

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3.2 Dual detection of MMPs

The internal impedance of an electrochemical biosensor can include charge-transfer

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resistance (Rct), film capacitance (Cf), and solution resistance (Rs). The equivalent circuit model is shown in Fig. S2 (Supplementary material). The diameter of the semicircle (real axis x-intercept) is highly relevant to interfacial charge-transfer resistance, if a Nyquist plot is drawn in the EIS assay. In order to analyze the influence of MMP concentration on impedance variation, impedance was measured in each chamber with MMPs of various concentrations. The data measured from chamber-III, when 1 pg/mL MMP7 was injected in the biochip, are shown in a Nyquist plot in Fig. 4. As reported by Wang et al. [40], the high frequency x-intercept impedance indicates the solution resistance. At low frequencies, the second real-axis intercept point represents the sum of Rs and Rct. The EIS fitting data are listed in Table S6 (Supplementary material). The first real-axis intercept of Rs varies little. On

ACCEPTED MANUSCRIPT the other hand, Rct is the smallest in the bare electrode state and largest in the modified electrode state. After reaction between the peptide and MMP, Rct is greater than that of the

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bare electrode state, and smaller than that of the modified electrode state. The film

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capacitance decreases after peptide immobilization, but it is nearly constant during the

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hydrolyzation reaction. This result suggests that only the charge-transfer resistance is involved in impedance change by the proteolytic digestion of peptide. The low frequency x-

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intercept value was utilized as analytical impedance. Impedance is increased because contact area between electrolyte and working electrode decreases if peptide is immobilized on the

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electrode surface. In addition, impedance is decreased because the amount of peptide covering the electrode surface is lessened, increasing ion access to the working electrode.

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Similarly, impedance decreases when a part of the peptide immobilized on the electrode

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surface is hydrolyzed by MMP and eliminated. As the concentration of MMP in the biochip

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increases, the peptide over the electrode surface will be hydrolyzed and eliminated much more. Therefore, the impedance reduction rate will also increase as current flowing into the electrode surface becomes smooth.

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In order to check whether identical impedance was measured under same experimental conditions, biochips were selected randomly from several wafers and we executed the following experiments. The intra-assay was conducted in 18 replicate experiments, and impedance was measured in cases of bare electrode and peptide-modified electrode (see Supplementary material Fig. S3). The coefficient of variation for the measured impedance was 1.03 in chamber-I, chamber-II, and chamber-III of the bare electrode, and was 0.95 in chamber-1 and chamber-III of the modified electrode. The large coefficient of variation for the bare electrode shows that variation of electrode resistance between the biochip is not small, although biochip electrodes were made by the same fabrication process. In addition, a nontrivial coefficient of variation at the peptide-modified electrode confirms that impedance

ACCEPTED MANUSCRIPT is not identical even though the amount of peptide fixed over the electrode is almost constant. Thus, MMP concentrations should be measured relative to the impedance variation rate or

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impedance reduction rate, relative to the impedance obtained after peptide immobilization, in

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order to consistently quantitate MMP concentrations despite chip variability.

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The impedance reduction rate used in this study is calculated as follows: impedance with post-enzymatic reaction electrode is subtracted from impedance with peptide modified

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electrode and then the result is normalized to the impedance of the modified electrode. The change in the low frequency x-intercept impedance of the Nyquist plot is normalized with

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respect to the modified electrode impedance value, which is measured before injection of the MMP sample. Impedance reduction rates for each biochip chamber were measured with

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MMP concentrations and plotted (Fig. 5). The measured data from chamber-I and chamber-

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III correspond to MMP2 and MMP7, respectively. The impedance reduction rates of MMP2

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and MMP7 show a growing trend as the MMP concentration increases. In chamber-II of the control, the impedance reduction rate was gauged as a constant value and was distinctly smaller than those of chamber-I and chamber-III. The measured values by peptides specific to

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MMP2 and MMP7 vary with the MMP concentration, but the value of the control is almost constant. This result shows that the concentration measurement of MMPs is performed correctly.

3.3 Assay performance of the multiplex biochip

Regression equations and adjusted R-squared values were found by nonlinear curve fitting of experimental data in Fig. 5, where the scale in x-axis is a log scale. The data show different patterns for MMP2 and MMP7. The peptide sequences differ, so the length of the cleaved peptides must be different between MMP2 and MMP7. This affects the impedance reduction

ACCEPTED MANUSCRIPT rate. The detection range of MMP2, which fits the calibration curve within a small margin of error (the impedance reduction rate is 8.68 – 56.34% and the adjusted R-squared value is

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0.957), is 0.05-200 ng/mL. The detection range of MMP7, on the other hand, is 0.0005-50

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ng/mL (the impedance reduction rate is 7.58 – 23.67% and the adjusted R-squared value is

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0.984). The lower detection limit is 0.5 pg/mL for both MMPs (the standard error of the impedance reduction rate is 0.88% and 1.54% for MMP2 and MMP7, respectively). Working

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ranges for MMP2 reported by other benchtop sensing operations are 0.1-1 g/mL [26] and 0.0005-50 ng/mL [22], and working ranges for MMP7 are 0.005-10 g/mL [18] and 0.01-5

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g/mL [19]. Therefore, the detection performance of the proposed biochip is high. To investigate the selectivity of the biochip, the impedance reduction rate of 50 ng/mL MMP7

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containing 1 mg/mL BSA and 1 mg/mL human hemoglobin was tested. Addition of BSA and

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hemoglobin into the enzyme sample resulted in signal loss of 16.67% and 21.3%, respectively,

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compared to the response of the biochip in MMP7 alone. Despite this signal suppression, however, the impedance reduction rates (19.72% and 18.63% in BSA and hemoglobin containing media, respectively) result in measurable changes (see Fig. 5). This result

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indicates, thus, MMP molecules can be detected in the presence of some other nonspecific proteins. For application in real samples, however, recalibration of the biochip should be carried out in the presence of nonspecific proteins found in blood. It takes 67 min 30 s to perform the assay using the biochip. This is very short compared with the 3-6 h required to perform traditional ELISA in the laboratory. The total amounts of peptide and enzyme solutions which are needed in the assay are very small, at to 10 and 20 l, respectively. Therefore, the developed biochip shows suitable performance as a POCT device.

4 Conclusions

ACCEPTED MANUSCRIPT We quantitatively measured concentrations of MMP2 and MMP7 using the change in impedance of the proposed biochip. The detection limits of both MMP2 and MMP7 were

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about 0.5 pg/mL. The sample volume required for sensing was reduced to about 20 l. Eventually, simultaneous diagnosis of ovarian and colorectal cancers may be possible by

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measuring biomarker concentrations and controls. MMPs are biomarkers of many cancers, and the developed biochip may serve as a microdevice which can diagnose several cancers as

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other MMP-related diseases early. Furthermore, the biochip designed in this study needs a short analysis time and small sample volume, and its simple structure makes mass production

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possible. Multiple biomarkers are present in the biochip; thus, in addition to simple target detection, the ratio between targets can be calculated. This study shows that POCT multiplex

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biodevice platforms for electrochemical measuring of two MMP biomarkers concurrently are

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feasible.

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There are some problems with the use of MMP as a biomarker of POCT biochip for cancer diagnosis. Concentrations of MMP2 and MMP7 measured from cancer patients vary greatly depending on the reported studies (see Supplementary material Table S1). Hence, it is

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hard to precisely determine the MMP cut-off values for diagnosis. When patient blood samples are used, diverse ingredients that may excluding MMPs can affect results. Biomarker can be affected by the surrounding environment too. The biochip must be distributed to people in order to be used as a POCT device, so the peptide may spoil in the process of distribution and storage. The biodevice will be more convenient to users if a single parameter is used instead of the impedance reduction rate. Further research is being carried out to resolve these problems. Future work will focus on producing a POCT device for practical application in the early detection of cancer. The MMP family can be used as biomarkers for various cancers [10], and peptides specific to each MMP are also able to be synthesized [32]. Moreover, the proposed method could be extended to other MMPs and other cancer diseases.

ACCEPTED MANUSCRIPT Furthermore, it is expected that the technique used in this study is not limited to MMP enzymes and could be applicable to most biomarkers of which findings of hydrolyzing

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activity is available.

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Conflict of interest

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There is no conflict of interest.

ACKNOWLEDGEMENT

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This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and

5 References

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Technology (2010-0010706)

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Figure captions

Fig. 1. Analytic concept for electrochemical MMP detection: (a) immobilization of peptide on an Au electrode; (b) measuring of impedance between electrodes after immobilization; (c)

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hydrolyzation of peptide by MMP; (d) re-measuring of impedance after enzyme reaction.

Fig. 2. Multiplex biochip: 2-D drawings of (a) the design, (b) the chaotic passive mixer

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(units: [mm]) and (c) a schematic sectional view of ‘A’, and a photograph of the fabricated microchip.

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Fig. 3. Cyclic voltammogram at the optimum conditions of 15 min immobilization, 1 min

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washing, and 30 min reaction, with a 1:1 mixture of 100 ng/mL MMP2 and 100 ng/mL

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MMp7 in the enzyme solution.

Fig. 4. Nyquist plot for electrodes in chamber-III when the concentration of MMP7 enzyme

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is 1 pg/mL.

Fig. 5. Impedance reduction rate as function of MMP concentration at each chamber in the biochip. The results of the control are plotted against MMP7 concentration. Detection data in the presence of 1 mg/mL BSA and 1 mg/mL hemoglobin is at 50 ng/mL MMP7.

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We propose a biosensor detecting MMP7 and MMP2 for cancer diagnosis.

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The multiplex biochip is a simple 2-electrode electrochemical POCT microdevice.

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Hydrolysis action with an unmodified peptide is adapted for MMP detection. Negative controls and a microfluidic mixer are used to enhance the performance.

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Concentrations of MMP2 and MMP7 are detected well concurrently.