Barcode DNA-Mediated Signal Amplifying Strategy for Ultrasensitive

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Barcode DNA-mediated Signal Amplifying Strategy for Ultrasensitive Biomolecular Detection on Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry Raheel Ahmad, Hyowon Jang, Bhagwan S. Batule, and Hyun Gyu Park Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01535 • Publication Date (Web): 05 Aug 2017 Downloaded from http://pubs.acs.org on August 6, 2017

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Analytical Chemistry

Barcode

DNA-mediated

Signal

Amplifying

Strategy

for

Ultrasensitive Biomolecular Detection on Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry

Raheel Ahmad#, Hyowon Jang#, Bhagwan S Batule and Hyun Gyu Park*

Department of Chemical and Biomolecular Engineering (BK 21+ program), KAIST, Daehak-ro 291, Yuseong-gu, Daejeon 34141, Republic of Korea

#

These authors contributed equally to this work.

*To whom correspondence should be addressed.

Correspondence: Hyun Gyu Park Ph.D., Department of Chemical and Biomolecular Engineering (BK21+ Program), KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea, Tel.: +82 42-350-3932, Fax: +82 42-350-3910, E-mail: [email protected] 1

ACS Paragon Plus Environment


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ABSTRACT We have devised a barcode DNA-mediated signal amplifying strategy for ultrasensitive biomolecular detection by utilizing matrix assisted laser desorption ionizationtime of flight mass spectrometry (MALDI-TOF MS). As a model target, thrombin was first captured by specific aptamer15 functionalized on magnetic beads (MBs-apt15) and sandwiched through the simultaneous interaction with gold nanoparticles modified with another specific aptamer29 and barcode DNA molecules (apt29-AuNPs-bcDNAs). The sandwiched complex was collected by convenient magnetic separation and then treated with potassium cyanide (KCN) to dissolve the gold nanoparticles (AuNPs) and consequently release the barcode DNA molecules (bcDNAs), which were then again magnetically separated and analyzed by using MALDI-TOF MS. Under optimized conditions, this strategy revealed an excellent sensitivity with a limit of detection of 0.89 aM in a wide linear detection range from 0 aM to 0.1 nM and exhibited an acceptable recovery for thrombin detection in complex biological matrices. This signal amplifying strategy based on MALDITOF MS could greatly enable the ultrasensitive detection of various low abundant biomolecules.

Keywords: Barcode DNA; MALDI-TOF MS; Thrombin; Aptamer 2


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1. INTRODUCTION Proteins play a vital role in many biological activities such as maintenance of body tissue, regulation of hormones, transportation and storage of certain molecules, and immune interaction, and thus the variation of protein expression level has served as an important indicator of specific biological status.1,2 Particularly, since the serious diseases like cancer or Alzheimer’s disease are often closely associated with an abnormal expression of an unique protein,3,4 sensitive and selective detection of proteins is highly demanded for many biological applications, including disease diagnoses and pharmaceutical development.5-9 Until now, tremendous methods have been developed to achieve ultrasensitive biomolecular detection based on fluorescence,10 colorimetry,11 electrochemical voltammetry,12 surface enhanced Raman spectroscopy (SERS),13 and surface plasmon resonance (SPR).14 While these methods have been proved to show quite good performance for the detection of proteins, their sensitivity is not high enough to detect some important cancer-related proteins due to their low abundance in body fluids or tissues. Therefore, there is great incentive still existing for the development of novel strategies enabling ultrasensitive detection of low abundant proteins. In order to satisfy this demand, polymerase chain reaction (PCR)-based methods such as liposome-PCR,15 immuno-PCR,16,17 aptamer-based affinity PCR,18-20 proximity ligation assay,21 immuno-rolling circle amplification,22 and T7 RNA polymerase-mediated amplification23 have been developed, which have achieved satisfactory limit of detection for low-abundant proteins. However, the requirements for thermal cycler,15 fluorophore labeling,20 and complex primer design22 have limited their wide spreads in practical applications. To overcome these limitations, nanomaterials such as gold nanoparticles 3



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(AuNPs), magnetic beads (MBs), nanowires, nanotubes, and quantum dots have been utilized by taking advantages of their unique features including large surface area and unique electrical, optical, and magnetic properties.24-34 Particularly, nanomaterial-coupled barcode studies have emerged as a promising amplification method for the detection of proteins or nucleic acids.35-42 In this method, the target molecules are sandwiched by two particle probes (typically, magnetic particles labeled with a target-specific ligand and AuNPs labeled with another target-specific ligand and barcode molecules), the complexes are collected through convenient magnetic separation, and then the barcode molecules are released and analyzed to identify the target molecule. Since even a single target molecule could attract the AuNP holding a large number of the barcode molecules, this method was very successfully applied to amplify the signal resulting from various target molecules.41 Meanwhile, mass spectrometry (MS) has attracted great attention as a promising analytical instrument for biomolecular detection, due to the several advantages such as easier automation and higher throughput and sensitivity over other analytical tools.43-45 Particularly, matrix assisted laser desorption ionization-time of flight (MALDI-TOF) MS coupled with soft ionization technique has significantly activated the development of the MS-based protein assays by enabling intact detection of protein or other large molecules.46,47 However, since these methods are not highly sensitive enough to detect low abundant proteins as well, the development of the MS-based signal amplifying strategies has been also quite challenging. Along this line, we herein describe a novel bio-barcode strategy to achieve the ultrasensitive detection of low abundant proteins based on MALDI-TOF MS. To achieve higher sensitivity over the previous MS-based bio-barcode assay,48 we introduced AuNP dissolution step to obtain more purified barcode molecules from lots of reagents involved in this assay and employed non-oxidized graphene nanosheets as a MALDI-TOF MS matrix by 4


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taking advantages of its high energy absorption ability and homogenous dispersion.49,50 To demonstrate the validity of this strategy, thrombin was chosen as a model target whose detection is very important for monitoring and assessing hemostasis and thrombosis.51-53

2. EXPERIMENTAL SECTION 2.1 Materials Gold(III) chloride trihydrate (HAuCl4.4H2O), tris (2-carboxyethyl) phosphine hydrochloride (TCEP), potassium cyanide (KCN), sodium citrate (Trisodium citrate dehydrate), tetrahydrofuran (THF), pyridine, n-methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO), human serum (from human male AB plasma), human serum albumin (HSA), bovine serum albumin (BSA), immunoglobulin A (IgA), lysozyme, and WhatmanTM qualitative filter paper (Grade1, circles, diam. 150 mm) were purchased from Sigma-Aldrich (USA). Dynabeads® M-270 streptavidin, protein-free blocking buffer, phosphate buffer saline (PBS, 1 M, pH 7.4), and ultraPure™ DNase/ RNase-Free distilled water were purchased from Thermo Fisher Scientific (USA). Graphite powder (SP-1 graphite powder) was purchased from Bay Carbon, Inc. (USA). Human Alpha Thrombin was purchased from Sekisui Diagnostics, LLC. (USA). 20 × SCC buffer was purchased from BIONEER (South Korea). All oligonucleotides (Table 1) used in this work were synthesized and purified by high-performance liquid chromatography (HPLC) by GENOTECH (Daejeon, South Korea). Sequence of bcDNA is designed to be included in range of 25-35 bases in length and not ended with multiple C or G residues which could cause non-specific interaction.54,55 OligoAnalyzer 3.1 program from IDT was used to check undesired non-specific interaction of bcDNA and aptamers. All other chemicals were of analytical grade and used without further purification. The washing buffer A consists of Tris-HCl (50 mM), NaCl (2 M), and Tween 20 (0.1%), pH 7.4. The washing buffer B consists of Tris-HCl (20 mM), KCl (5 mM), MgCl2 (1 mM), NaCl (140 mM), CaCl2 (1 mM), and Tween 20 (0.1%), pH 7.4. The storage buffer 5



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consists of Tris-HCl (20 mM), CaCl2 (1 mM), MgCl2 (1 mM), KCl (5 mM), NaCl (140 mM), and ethylene diamine tetraacetic acid (EDTA, 1 mM), pH 8.0. The immobilization buffer consists of PBS (10 mM), EDTA (1 mM), and NaCl (0.6 M).

2.2 Instruments The morphologies of AuNPs and DNA-functionalized AuNPs were examined by transmission electron microscope (TEM, Titan Double Cs corrected, FEI, USA). The characterization for monolayer of non-oxidized graphene nanosheets was carried out by high resolution mode of field emission transmission electron microscope (FE-TEM, FEI, USA). The interlayer spacing (d-spacing) and diffraction angle (002 peak) of non-oxidized graphene nanosheets were determined by high resolution powder x-ray diffractometer (HR PowderXRD, RIGAKU, USA). All fluorescence analyses were carried out by Tecan Infinite M200 pro microplate reader (TECAN, Switzerland). The loading amount of DNA on AuNPs was calculated by using nanodrop spectrophotometer (ND-1000, USA). The mass spectrums of the barcode DNA molecules (bcDNAs) were analyzed by MALDI-TOF MS (Bruker Daltonics Inc., Germany).

2.3 Preparation of magnetic beads functionalized with aptamer15 (MBs-apt15) Streptavidin-coated MBs were magnetically separated from 600 µL of 1 mg/mL Dynabeads® M-270 streptavidin solution. The collected MBs were rinsed twice with both washing buffer A and 0.5 × SSC buffer and re-dispersed in 300 µL of washing buffer A. 150 µL of 10 µM biotin-modified aptamer15 (apt15) solution was added to the above solution and incubated at 37 °C for 1.5 h for the complete conjugation of apt15 on MBs. The resulting 6


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MBs modified with apt15 (MBs-apt15) were magnetically separated and rinsed twice with washing buffer B, which were then re-suspended in 300 µL of protein free blocking buffer followed by incubation at 4 °C for 2.5 h to minimize the non-specific binding. The MBsapt15 were next rinsed twice with storage buffer and finally re-dispersed in 300 µL of storage buffer. The as-prepared MBs-apt15 were stored at 4 °C before use.

2.4 Preparation of AuNPs and their modification with aptamer29 and bcDNAs (apt29-AuNPs-bcDNAs) Citrate stabilized AuNPs, with an average diameter of 50 nm, were synthesized according to previously reported protocol with a slight modification.56 All glassware were thoroughly cleaned with aqua regia (HNO3/HCl, 1:3, v/v) followed by washing twice with water and drying for use. 200 mL of 0.4 mM aqueous gold (III) chloride tri-hydrate (HAuCl4.4H2O) solution was heated to boil and then 2 mL of 38 mM sodium citrate (Trisodium citrate dehydrate) was quickly added to reflux under stir. After the color of the solution changed to wine red, the solution was further heated for 35-40 min and then allowed to cool down at room temperature. The solution was finally filtered through filter (WhatmanTM qualitative filter paper) and the concentration of the AuNP solution was calculated by following the previously reported protocol,57 which was then stored at 4 °C before use. For the coupling of the bcDNAs and aptamer29 (apt29) on AuNPs, 6 µL of 100 µM bcDNAs, 6 µL of 10 µM apt29 and 6 µL of 10 mM TCEP were mixed, and distilled water was added to make a final volume of 60 µL, which was then incubated at room temperature for 2 h to reduce the disulfide bond. The solution (molar ratio of the bcDNAs: apt29 = 10:1) 7



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was again mixed with 6 µL of 50 mM TCEP and TE buffer was added to make a final volume of 80 µL. The solution was incubated at 30 °C for 1.5 h to reduce the leftover disulfide bond, and 500 µL of 0.25 nM AuNP solution, 110 µL of 50 mM PBS, and 10 µL of 0.1 M EDTA were sequentially added, which was incubated at 30 °C for 5 h. Then, 30 µL of 2 M NaCl in 0.01 M PBS was added into the reaction mixture with 30 min of time interval to make a final NaCl concentration of 0.6 M.58 Finally, the mixture was incubated at 30 °C for 16 h. After centrifugation at 14000 rpm for 20 min, the AuNPs were carefully collected by removing the supernatant. The collected AuNPs were re-dispersed in 200 µL immobilization buffer. The centrifugation and re-dispersion were repeated twice for the complete removal of the unreacted bcDNAs and apt29. The resulting apt29-AuNPs-bcDNAs were stored at 4 °C before use.

2.5 Formation of the sandwiched complex of MBs and AuNPs (MBsapt15/thrombin/apt29-AuNPs-bcDNAs) through the interaction with thrombin and separation of bcDNAs from the sandwiched complex In a typical experiment, 200 µL of thrombin at different concentrations were added into 20 µl of MBs-apt15. The mixture was then incubated at 37 °C for 2 h on a dry shaker (200 rpm) to ensure the complete interaction and capture of thrombin. Afterwards, the MBsapt15 with the captured thrombin were magnetically separated and rinsed three times with 200 µL of washing buffer B. The resulting MBs-apt15 with the captured thrombin were mixed with 100 µL of apt29-AuNPs-bcDNAs solution. The mixture was then incubated at 37 °C for 90 min under 8


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gentle shaking, thereby forming the MBs-apt15/thrombin/apt29-AuNPs-bcDNAs sandwiched complex. The sandwiched complex was next magnetically separated and carefully rinsed three times with 100 µL of washing buffer to remove the unsandwiched apt29-AuNPsbcDNAs, which was finally re-suspended in 20 µL of washing buffer. The resulting sandwiched complex (MBs-apt15/thrombin/apt29-AuNPs-bcDNAs) was incubated for 1 min under heating after mixed with KCN (final concentration, 50 mM), which resulted in the dissolution of AuNPs and release of bcDNAs. Finally, the magnetic separation was again applied to efficiently recover the bcDNAs from the sandwiched complex.

2.6 MALDI-TOF MS analysis Non-oxidized graphene nanosheets were synthesized by following the previously reported protocol.49,50 One milligram of non-oxidized graphene powder was dispersed in 1 mL of distilled water and sonicated for 15 min to make a homogenous suspension. About 1 µL of suspension (matrix) was pipetted onto the MALDI plate and left at room temperature for 10-15 min to evaporate water, which led to the formation of crystal structure on the target spot. Next, 1µL of analyte solution was embedded on the crystal matrix and left at room temperature to evaporate water. The MALDI-TOF MS analysis was finally performed by employing the mass spectrometer with laser (Nd-YAG) operated at 355 nm. The voltage, delayed extraction time, grid voltage, laser rate, and laser shots were set as 20 KV, 220 ns, 87.8%, 1~200 Hz, and 500, respectively.

9



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3. RESULTS AND DISCUSSION 3.1 Design of strategy As illustrated in Scheme 1, AuNPs were first modified with bcDNAs as an unique identification tag for target molecule and thrombin binding apt29 which would be recognized by the thrombin binding site II (Exosite II).59 We next modified MBs with biotin-labelled another thrombin aptamer known as apt15, which would be recognized by the thrombin binding site I (Exosite I).59 In the presence of thrombin, the interaction of thrombin with the two specific aptamers on AuNPs and MBs would lead to the formation of the sandwiched complex (MBs-apt15/thrombin/apt29-AuNPs-bcDNAs). The sandwiched complex was then magnetically separated and treated with KCN to dissolve AuNPs, consequently releasing the bcDNAs from the sandwiched complex. The released bcDNAs were conveniently collected through magnetic separation and analyzed by MALDI-TOF MS in which non-oxidized graphene nanosheets were used as a matrix. The non-oxidized graphene nanosheet matrix employed in this work possesses hydrophobic nature yielding high surface area which is quite advantageous for the biomolecular detection. Furthermore, it could greatly simplify sample preparation, eliminate interference from background matrix ion, and trap analytes by pi-pi interaction and hydrophobic interaction, which leads to the enhanced limit of detection and 10


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high reproducibility.50,60,61

3.2 Characterization of DNA-modified nanoparticles and graphene nanosheet matrix The unmodified AuNPs and DNA-modified AuNPs (apt29-AuNPs-bcDNAs) were characterized by using transmission electron microscope (TEM). According to the previous report62 that the number of DNA strand per AuNP significantly increased as the size of AuNPs increased from 20 to 50 nm, 50 nm AuNPs were employed in this work (Figure 1A). Figure 1B shows the morphology of apt29-AuNPs-bcDNAs, which revealed the significantly increased average diameter of AuNPs (70 nm) confirming the successful modification of bcDNAs and apt29 on AuNPs. The size distributions of the unmodified AuNPs and apt29AuNPs-bcDNAs were obtained by Image J software (inset in Figure 1A and 1B). UV-vis spectra were analyzed to further characterize the unmodified AuNPs and apt29-AuNPs-bcDNAs. The unmodified AuNPs showed an obvious absorbance peak at 534 nm, and a 4 nm red shift associated with the localized surface plasmon resonance effect63 was observed from apt29-AuNPs-bcDNAs, which again indicated that bcDNAs and apt29 were successfully conjugated on AuNPs (Figure 1C). MBs and MBs-apt15 were also analyzed by UV-vis spectra after they were treated with SYBR green II specifically binding to ssDNA (Figure 1D).64 A strong emission peak of SYBR green II at 522 nm was observed from MBsapt15 confirming the successful conjugation of apt15 on MBs. The non-oxidized graphene nanosheets were characterized through high resolution mode of transmission electron microscope (HR-TEM) analysis (Supporting Information, Figure S-1), which indicated that the non-oxidized graphene nanosheets have monolayer structure and a shape like crumpled silk waves.49 The monolayered nanosheet structure of 11



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non-oxidized graphene was further confirmed by determining the diffraction angle (002 peak, 2θ = 26.7°) and layer-to-layer distance (d-spacing, 0.335 nm) through x-ray diffraction (XRD) analysis (Supporting Information, Figure S-2).65,66 The monolayer characteristics of nonoxidized graphene nanosheets enables homogeneous dispersion and yields high surface area, which would lead to high energy absorption ability of matrix and consequently enhanced signal intensity from MALDI-TOF MS analysis.60

3.3 Ultrasensitive detection of thrombin We first optimized the concentration of potassium cyanide (KCN) to sufficiently dissolve the AuNPs for the collection of the bcDNAs from the sandwiched complex. As presented in Figure S-3A (Supporting Information), the absorbance at 534 nm corresponding to AuNPs decreased with the increased concentration of KCN and completely disappeared at KCN (initial concentration, 100 mM) indicating that almost all the AuNPs were dissolved, which was employed in further experiments. We then conducted polyacrylamide gel electrophoresis (PAGE, 16%) to analyze the bcDNAs collected from the sandwiched complex through AuNP dissolution by KCN at different concentrations. As expected, KCN at the higher concentration resulted in the more intensified band corresponding to the bcDNAs (Supporting Information, Figure S-3B) indicating that KCN at the higher concentration is the more efficient to release the bcDNAs from the sandwiched complex. The band corresponding to apt29 was also observed on the higher position of the PAGE gel whose intensity was lower than that for bcDNAs and also correctly increased with the increased concentration of KCN. Next, to prove the feasibility of this strategy, a sample containing thrombin (1 nM) was subjected to the assay procedure and the bcDNAs produced from the assay were analyzed by PAGE and MALDI-TOF MS, which were then compared with a negative control sample w/o 12


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thrombin. As a result shown in Figure S-4 (Supporting Information), both the clear gel band and MALDI-TOF MS peak corresponding to bcDNAs were successfully observed only from the sample containing thrombin, while there was no signal for bcDNAs detected from the sample w/o thrombin. The mass peak was correctly observed at 8450.7 m/z, which is identical with the molecular weight of the bcDNAs. All these results clearly prove the feasibility of this novel strategy. We then applied this strategy for the detection of thrombin by employing the samples containing thrombin at different concentrations and the bcDNAs produced from the assay were analyzed by PAGE and MALDI-TOF MS. As a result presented in Figure 2A and 2B, both of the gel band and mass peak intensities correctly increased with the increase of thrombin concentration. When the normalized mass peak intensity (%) was plotted with thrombin concentration in Figure 2C, this strategy exhibited excellent linear relationship with thrombin concentration in a range from 0 aM to 0.1 nM. The regression equation was y = 12.659x – 2.1239 where y is the normalized mass peak intensity and x is thrombin concentration with a correlation coefficient of 0.9834. The limit of detection was determined to be 0.89 aM, which is the best among those of recently reported strategies (Supporting Information, Table S-1). To demonstrate the specificity of this strategy, several non-target proteins including human serum albumin (HSA, 100 nM), lysozyme (100 nM), immunoglobulin G (IgG, 100 nM), and bovine serum albumin (BSA, 100nM) were subjected to the assay procedure and the bcDNAs produced from the assay were analyzed by PAGE and MALDI-TOF MS. As shown in Figure 3A and 3B, neither PAGE nor MALDI-TOF MS analysis showed any significant signal corresponding to the bcDNAs for all the tested non-target proteins even though they were applied at 100-fold higher concentration. This observation clearly confirms 13



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the excellent specificity of this method only towards the target thrombin. To finally verify the practical capability of this strategy for complex biological samples, we applied this assay to detect thrombin spiked in human serum. The human serum was isolated from blood sample, diluted in a 1:10 ratio with distilled water, and spiked with thrombin at a concentration of 1 nM. The MALDI-TOF MS response obtained from thrombin spiked in the human serum was almost same with that from thrombin in a simple buffer solution (Figure 4), which finds its potential application in complex biological matrices

4. CONCLUSIONS In conclusion, ultrasensitive protein assay strategy, capable of an attomolar detection, has been developed based on the bcDNA-mediated signal amplification coupled with MALDI-TOF MS. By modifying each AuNP to carry approximately thousands of the bcDNAs for a single aptamer DNA molecule, the MS response resulting from the single interaction between model target thrombin and its aptamer was greatly amplified. Consequently, we were able to achieve an extremely low limit of detection of 0.89 aM in a wide linear detection range from 0 aM to 0.1 nM with the high specificity towards the target thrombin. All these results proves the excellent capability of this strategy to reliably and very sensitively monitor low-abundant proteins and this approach could serve as a promising platform for the development of various novel signal-amplifying strategies for the ultrasensitive detection of various biomolecules.

ACKNOWLEDGMENTS 14


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This work was financially supported by the Center for BioNano Health-Guard funded by the Ministry of Science, ICT and Future Planning (MSIP) of Korea as a Global Frontier Project (Grant H-GUARD-2013M3A6B2078964) and by Basic Science Research Program

through

the

NRF

funded

by

the

Ministry

of

Education

[No.

2015R1A2A1A01005393].

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publication website at DOI: x.xxxx/ Characterization of non-oxidized graphene nanosheets by TEM and XRD analysis, effect of KCN concentration on the dissolution of AuNPs, feasibility test result of the sensing strategy and comparison of various strategies for their performance to detect thrombin.

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Table of Content (TOC)

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Scheme 1. Schematic Illustration of the bcDNA-mediated signal amplifying MALDI-TOF MS for thrombin detection.

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Figure 1. (A, B) TEM images of unmodified AuNPs and apt29-AuNPs-bcDNAs (Inset: size distribution), (C) UV-vis spectra of unmodified AuNPs and apt29-AuNPs-bcDNAs, (D) Emission spectra of SYBR green II from unmodified MBs and MBs-apt15.

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Figure 2. Sensitivity of the bcDNA-mediated signal amplifying MALDI-TOF MS assay for thrombin detection. (A) PAGE image of bcDNAs collected from the assay of thrombin at different concentrations. Lane 1, apt29 (control); Lane 2, bcDNA (control); Lane 3, 0.1 nM; Lane 4, 0.01 nM; Lane 5, 1 pM; Lane 6, 0.1 pM; Lane 7, 10 fM; Lane 8, 1 fM; Lane 9, 100 aM; and Lane 10, w/o thrombin (0 aM). (B) MALDI-TOF MS response at 8450.7 m/z from bcDNAs collected from the assay of thrombin at different concentrations. (a) 0.1 nM, (b) 0.01 nM, (c) 1 pM, (d) 0.1 pM, (e) 10 fM, (f) 1 fM, (g) 100 aM, and (h) 0 aM. (C) The linear relationship between thrombin concentration and MALDI-TOF MS peak intensity in a range from 0 aM to 0.1 nM. The peak intensity at 8450.7 m/z obtained from the sample containing thrombin (0.1 nM) was normalized as 100 %. The Error bar shows the standard deviation of the three independent measurements for each sample. 21



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Figure 3. Specificity of the bcDNA-mediated signal amplifying MALDI-TOF MS assay for thrombin detection. (A) PAGE image of bcDNAs collected from the assay of target thrombin and other non-target proteins. Lane 1, aptamer29 (control); Lane 2, bcDNA (control); Lane 3, thrombin (1 nM); Lane 4, blank; Lane 5, HSA (100 nM); Lane 6, Lysozyme (100 nM); Lane 7, IgG (100 nM); and Lane 8, BSA (100 nM). (B) MALDI-TOF MS response from bcDNAs collected from the assay of target thrombin and other non-target proteins. (a) Thrombin (1 nM), (b) distilled water (blank), (c) HSA (100 nM), (d) lysozyme (100 nM), (e) IgG (100 nM), and (f) BSA (100 nM). (C) Normalized mass peak intensity (%) obtained from thrombin and other non-target proteins. The peak intensity at 8450.7 m/z obtained from the sample containing thrombin (1 nM) was normalized as 100 %. Error bar shows the standard deviation of the three independent measurements of each sample. 22


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100 90 Normalized mass peak intensity (%)

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80 70 60 50 40 30 20 10 0 Control

Serum

Thrombin

Thrombin+Serum

Figure 4. MALDI-TOF MS response from bcDNAs collected from the assay of thrombin spiked in human serum. (1) Distilled water w/o thrombin (control), (2) human serum (10%) w/o thrombin, (3) thrombin (0.1 nM) in distilled water, and (4) thrombin (0.1 nM) spiked in human serum (10%). The peak intensity at 8450.7 m/z obtained from the thrombin (0.1 nM) spiked in human serum sample was normalized as 100 %.

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Probe

Sequence (5ʹ

3ʹ)

Barcode DNA

GAA ATG AAG GAG -A15-(C3)Thiol

Aptamer15

Biotin-A15-GGT TGG TGT GGT TGG

Aptamer29

Thiol(C6)-A15- AGT CCG TGG TAG GGC AGG TTG GGG TGA CT

Table 1. Sequences of oligonucleotides used in this study

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Barcode DNA-Mediated Signal Amplifying Strategy for Ultrasensitive

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