Multiplexed Detection of Tumor Markers with Multicolor Polymer Dot

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Multiplexed Detection of Tumor Markers with Multicolor Polymer Dot-Based Immunochromatography Test Strip Chia-Chia Fang, Chia-Cheng Chou, Yong-Quan Yang, WeiKai Tsai, Yeng-Tseng Wang, and Yang-Hsiang Chan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04411 • Publication Date (Web): 29 Dec 2017 Downloaded from http://pubs.acs.org on December 30, 2017

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Analytical Chemistry

Multiplexed Detection of Tumor Markers with Multicolor Polymer Dot-Based Immunochromatography Test Strip Chia-Chia Fang,† Chia-Cheng Chou,† Yong-Quan Yang,† Tsai, Wei-Kai,† Yeng-Tseng Wang,‡ and Yang-Hsiang Chan*† † ‡

Department of Chemistry, National Sun Yat-sen University, 70 Lien Hai Road, Kaohsiung, Taiwan 80424 Department of Biochemistry, Kaohsiung Medical University, 100 Tzyou 1st Road, Kaohsiung Taiwan 80708

ABSTRACT: There have been ongoing efforts to develop more sensitive and fast quantitative screening of cancer markers by use of fluorometric immunochromatographic test strip (ICTS) since the remarkable advances in fluorescent nanomaterials. Semiconducting polymer dots (Pdots) have recently emerged as a new type of biocompatible fluorescent probes with extraordinary brightness which is suitable for biological and clinical use. Here we developed Pdot-based ICTS for quantitative rapid screening of prostate-specific antigen (PSA), alpha-fetoprotein (AFP), and carcinoembryonic antigen (CEA) in 10 min. By taking advantage of the ultrahigh fluorescence brightness of Pdots, this immunosensor enabled much better detection sensitivity (2.05, 3.30, and 4.92 pg/mL for PSA, AFP, and CEA, respectively), which is at least 2 orders of magnitude lower than that of conventional fluorometric ICTS. Furthermore, we performed proof-of-concept experiments for simultaneous determination of multiple tumor markers in a single test strip. These results demonstrated that this Pdot-based ICTS platform is a promising candidate for developing new generations of point-of-care diagnostics. To the best of our knowledge, this is the first example of Pdot-based ICTS with multiplexing capability.

INTRODUCTION

beneficial for sensitive quantification of analytes with small sample volumes.19 Among these fluorescent labels, inorganic quantum dots appear to be one of the promising candidates because of their high quantum yields, large extinction coefficients, and narrow-band emissions.20 Despite these impressive optical properties of quantum dots, there are several limitations for their practical use. For example, the toxicity of the inorganic elements is detrimental to the environment and the interactions to metal ions or biothiols in physiological fluids might affect their optical properties. 21-24 Therefore, selecting a biocompatible, chemically stable, ultrabright, and photostable fluorescent probe for fluorometric ICTS is highly desired.

Point-of-care (POC) diagnostics has become an important in vitro clinical medicine subject which is particularly suitable for on-site patient care because POC tests do not require sophisticated instruments or highly skilled laboratory staffs to obtain the result in a timely manner.1,2 Moreover, POC approaches are very cost-effective, easy-to-use, highthroughput capable, and fast-responsive, which greatly favors the rapid disease screening and immediate clinical-medical treatment. Immunochromatographic test strip (ICTS) or lateral flow immunoassay, introduced by Unipath in late 1900s,3 is the most commonly diagnostic format of POC and has been extensively used for medical diagnosis, therapeutic monitoring, environmental analysis, food safety, forensic science, and security screening.1,2,4-11

Semiconducting polymer dots (Pdots) have recently emerged as a new class ultrabright fluorescent probe for biological and sensing applications, as well as material science.25-44 There are several advantages for Pdots to be employed in ICTS: i) The super high fluorescence brightness that can provide good detection sensitivity with low background interference; ii) High colloidal and photo-stability in physiological conditions, the commonly used environment of ICTS; iii) Multicolor emissions from different types of Pdots with single excitation wavelength, allowing for multiplexed detection; iv) Facile surface functionalization for further conjugation with diverse biomolecules to specifically target analytes of interest.

In ICTS, signal reporting reagents play an important role in the detection sensitivity and selectivity. To date, most ICTS is based on the colorimetric method in which colloidal gold nanoparticles are widely adopted in this system owing to their prominent colors from surface plasmon resonance. The qualitative or semiquantitative detection results can be directly visualized and then analyzed by naked eyes. However, the detection limit of colorimetry-based ICTS remains to be improved for the detection of trace analytes because subtle changes of analyte concentration might not be reflected from simply the shades of signal reporters. Therefore, there have been great efforts to promote the detection sensitivity of ICTS by employment of various alternative labels with fluorescent property, including organic dyes, carbon dots, up-conversion nanocrystals, quantum dots, and dye-doped nanoparticles.4,12-18 As compared to colorimetric ICTS, fluorometric ICTS possesses advantageous features of high sensitivity, high contrast, and low background interference, which are

In this study, we report the first example of Pdot-based ICTS for the detection of tumor markers simultaneously with trace sample volume. Three tumor markers, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and prostate-specific antigen (PSA), were used as models to demonstrate the qualitative and quantitative performance of the Pdot-based ICTS. Specifically, we fabricated a control line (modified by

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capture antibody) and a test line (modified by IgG antibody) on a single test strip and the concentration of the target antigen could be measured by the fluorescence ratio of test line to control line. This Pdot-based ICTS platform was thereon utilized to rapidly determine the concentrations of tumor markers in human serum samples. More importantly, three different types of Pdots of three distinct emissions were functionalized with different antibodies and then fabricated onto the same conjugation pad. This design allows the simultaneous detection of multiple tumor markers in a single test strip. This new type of Pdot-based ICTS may open a new avenue for rapid and accurate point-of-care cancer diagnostics.

antibody-conjugated Pdots were stored at 4 °C in the dark and remained stable for at least 2 months. Preparation of Immunochromatography Test Strip Based on Pdot-antibody Probes. First, the freshly prepared 4 mL of Pdot-antibody solution was concentrated by a centrifugal ultrafiltration tube (Pall Microsep Advance PES Omega, MWCO: 100 kDa) to 800 L at 1600 rpm for 4 min. The test strip is made of a sample pad, conjugate pad, absorbent pad, and nitrocellulose membrane. The sample pad was used as received without further modification. The conjugate pad was prepared by immersing into the Pdot solution, containing 1 L concentrated Pdot-antibody probes, 49 L pure water, 2 L 5% (w/w) Triton X-100, 2 L 5% (w/w) PEG400, 4 L 5% (w/w) sucrose, 1 L 1M HEPES buffer, and 2.5 L (w/w) ethylene glycol. The conjugate pad along with the mixture solution was allowed to dry under vacuum for 1 h. The nitrocellulose membrane was cut to 4 mm in width, and the test and control lines were fabricated by the fountain pens loaded with PSA/AFP/CEA antibody (1.5 g/mL) solutions and IgG secondary antibody (0.5 g/mL) solutions, respectively. The nitrocellulose membrane was further dried under vacuum for 15 min. After that, the test strip was assembled by adhering the absorbent pad, conjugate pad, and sample pad onto the nitrocellulose membrane, sequentially. Finally, the test strip was fitted into the 4 mm plastic cassette.

EXPERIMENTAL SECTION Chemicals. All reagents were purchased from SigmaAldrich or Alfa Asear and used as received unless indicated elsewhere. Polystyrene graft ethylene oxide functionalized with carboxylic end group (PS-PEG-COOH, Mn = 6500 Da of PS moiety; 4600 Da of PEG-COOH; polydispersity, 1.3) was purchased from Polymer Source, Inc. (Dorval PQ, Canada) and used as received. CEA, AFP, IgG, and PSA proteins or antibodies were all purchased from Fitzgerald Industries International, Inc. (Concord, MA). Mouse anti-human CEA antibodies (catalog number: 10-7882 and 10-7882) were modified onto the test line and the Pdot surface, respectively. Mouse anti-human PSA antibodies (catalog number: 10-P20E and 10-P20D) were modified onto the test line and the Pdot surface, respectively. Rabbit anti-human AFP (catalog number: 70R-10676) and mouse anti-human AFP (catalog number: 10-A100B) were modified onto the test line and the Pdot surface, respectively. Chicken anti-mouse IgG (H+L) secondary antibody (41R-1047) was modified onto the control line. CEA antigen (30-AC26), PSA antigen (30C-CP1017U), and AFP antigen (30-1029) were diluted into the desired concentrations in PBS buffer solutions in the experiments. High purity water (18.2 M•cm) was used throughout the experiment. Nitrocellulose membranes (8 m, CNPC), sample pads (GFB-R4), conjugate release matrices (PT-R5), and absorbent pads (AP080) were all purchased from Advanced Microdevices Pvt. Ltd. (India) and then cut into appropriate sizes to assemble to test strips, which were fit into 4 mm plastic cassettes.

Immunodetection of PSA/AFP/CEA Pdot-Based ICTS. The standard samples were prepared by the addition of 0-20 L PSA/AFP/CEA (0.1 g/mL), 40 L 5% (w/w) PEG400, 4 L serum, and 40-60 L 20 mM HEPES together. For real samples, 60 L serum was spiked with PSA/AFP/CEA and then added with 40 L 5% PEG400. The above samples were added into the sample port on the ICTS and waited for 10 min to read the results. The fluorescence images of test strips were photographed by a Nikon D5500 digital camera under 365 nm UV light and then processed using ImageJ 1.51 for analyzing the fluorescence intensity of the control and test lines. For each concentration, 5 replicate measurements were carried out. Characterization of Pdots. The average particle size was determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM). TEM images of the synthesized Pdots were acquired using a JEOL 2100 transmission electron microscope at an acceleration voltage of 200 kV. For TEM, 10 L of Pdot aqueous solution (diluted by 20 times) was placed onto a carbon-coated grid and allowed for water evaporation at room temperature. The DLS data of Pdots in aqueous solution were acquired by dynamic light scattering system (Malvern Zetasizer Nano S). The UV-visible absorption spectra of Pdots were characterized on a Dynamica Halo DB20S spectrophotometer. The fluorescence spectra were measured by use of a Hitachi F-7000 ﬂuorometer (Hitachi, Tokyo, Japan) under 405 nm excitation. For gel electrophoresis experiments, the gel was prepared by mixing 0.2 g agarose and 30 mL water together and then put into a microwave oven for heating 2-3 min until the solution became clear. After that, the solution was added 10 L of 1 M HEPES and 1.2 mL of 5% PEG400 and the mixtures were poured into the electrophoresis channel mold. The Pdot samples (70 L) were then loaded into the channels with the aid of 30 L glycerol and ran in 20 mM HEPES buffer under an applied voltage of 100 V for 30 min.

Preparation of Pdots. First, 200 L of PF-TC6FQ, PFCN, or PFO (1 mg/mL in THF), and 20 L of PS-PEG-COOH (2 mg/mL in THF) were mixed together in 5 mL of THF. The mixture was sonicated for 15 s and then injected into 10 mL of H2O under violent sonication. After that, THF was removed by purging with dry N2 on a 70 °C hot plate for 25 min. The resulting Pdot solution was cooled down to room temperature and then filtered through a 0.22 m cellulose acetate syringe filter to remove any possible particle aggregates. Antibody Conjugation of Pdots. 4 mL of Pdot solution was added into a 20 mL of vial, and then 80 L of 1M HEPES buffer, 80 L of 5% PEG was added into the vial. 120 L of PSA (0.1 g/mL) or 60 L of AFP (0.1 g/mL) or 20 L of CEA (0.1 g/mL) antibody was added into the mixture. After that, 80 L of fresh-prepared 1-Ethyl-3-(3dimethylaminopropyl)carbodiimide (5 g/mL) and 20 L of nhydroxysuccinimide (5 g/mL) was added and then stirred for 4 h at room temperature. After the reaction, the Pdot-antibody bioconjugates were purified by gel filtration using Sephacryl HR-300 gel media to remove the unreacted antibodies. The


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O C N H

COOH

Scheme 1. Schematic Showing the Preparation of Pdot-Based Immunochromatographic Test Stripa

NH2

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aFirst,

semiconducting polymer PF-TC6FQ/PFCN/PFO and carboxyl-functionalized polystyrene PS-PEG-COOH were mixed well in THF and then coprecipitated in water under vigorous sonication to form carboxyl-functionalized Pdots. The Pdots were subsequently conjugated with PSA/AFP/CEA antibody and then loaded onto the conjugate pad. The nitrocellulose membrane was fabricated with the control line and the test line and then assembled with the absorbent pad, conjugate pad, and sample pad in sequence. The result interpretation was carried out by comparing the fluorescence intensity of the control line and the test line. Figure 1. (A) Absorption spectra of PF-TC6FQ, PFCN, and PFO Pdots in aqueous solutions. The inset shows the photograph of the Pdot solutions under ambient light. (from left to right: PF-TC6FQ, PFCN, and PFO Pdots). (B) Emission spectra of PF-TC6FQ, PFCN, and PFO Pdots in aqueous solutions. The inset shows the photograph of the Pdot solutions under 365 nm UV light. (from left to right: PF-TC6FQ, PFCN, and PFO Pdots). (C) Hydrodynamic diameters of PF-TC6FQ (left), PFCN (middle), and PFO (right) Pdots measured by dynamic light scattering. The upper panels represent the data of as-prepared Pdots while the bottom panels represent that of antibody-modified Pdots (i.e., PFTC6FQ-PSA, PFCN-AFP, and PFO-CEA). The insets in the upper panels show their corresponding transmission electron microscopy images. The scar bars are 100 nm. (D) Gel electrophoresis of bare Pdots and antibody-functionalized Pdots.

RESULTS AND DISCUSSION Our aim was to design multiplex lateral flow immunoassay for detection of tumor biomarkers with multicolor Pdots. Here we selected three different semiconducting polymers, PF-TC6FQ, PFCN, and PFO, because they have distinct emission colors (red, green, and blue, respectively) under the same excitation light (i.e., 365 nm light in this work). This would allow us to develop multiplexed ICTS based on multicolor Pdots for simultaneous detection of tumor markers on a single test strip. A B

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Preparation of Pdot-Antibody Conjugates. As shown in Scheme 1, multicolor Pdots were prepared by coprecipitating with carboxyl-functionalized polystyrene (i.e. PS-PEG-COOH) to

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form carboxylic acid functional Pdots for further antibody conjugation. The absorption and emission spectra of the three

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different types of Pdots were shown in Figure 1A,B, respectively. Due to their broad and strong absorption in the UV-visible regions, three distinct emission colors (blue, grren, and red) of these Pdots could be easily discriminated under commonly used 365 or 405 nm light excitation. This feature is advantageous for the following design of multiplexed ICTS on the basis of these Pdots. Once the Pdots were ready, we decorated PSA, AFP, and CEA antibodies onto the surfaces of PF-TC6FQ, PFCN, and an PFO Pdots, respectively. As

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Analytical Chemistry displayed in Figure 1C, the average hydrodynamic diameters of the antibody-conjugated Pdots increased from 21, 18 and 21 nm to 24, 22, and 35 nm for PF-TC6FQ (PSA), PFCN (AFP), and PFO (CEA) Pdots, respectively. We found that the hydrodynamic sizes of Pdots increased by 3-4 nm after PSA/AFP surface functionalization. It is probably due to the large molecular weight of PSA (29 kDa) or AFP (69 kDa), which is consistent with the result observed by Chiu’s group (~2 nm increase after streptavidin bioconjugation).45 The relatively large molecular of CEA ( 180 kDa) caused larger size increase for PFO Pdots after CEA conjugation. We also performed gel electrophoresis to further verify the formation of functional groups on the Pdot surface. Figure 1D shows that the bare (carboxyl-functionalized) Pdots displayed a higher mobility as compared to that of antibody-modified Pdots. The slower mobility of antibody-conjugated Pdots could be attributed to the decrease in the negative surface charges along with the increased particle sizes, suggesting the successful antibody functionalization on the surface of Pdots.

positive result. On the other hand, the lack of target antigen in the samples resulted in a negative result, in which no fluorescence from Pdots in the test zone could be observed. In both cases (with and w/o antigen), Pdot-antibody conjugates were bound to the control line by bare IgG. The results from the test strip could be interpreted directly by naked eyes under hand-held UV lamp excitation. The quantitative concentrations of the analytes in the samples could be further determined by the fluorescence intensity ratios of the test line to the control line (T/C). We also evaluated the specificity of this Pdot-based ICTS. In this experiment, we fabricated PSA antibodies and PdotPSA conjugates onto the test zone and the conjugate pad of the test strip, respectively. We then used samples containing PSA, AFP, CEA, fetal bovine serum (FBS), or RPMI-1640 medium to test the non-specific interference. As displayed in Figure 2A, we could clearly observe that only PSA was specifically bound to the test line and recognized by Pdot-PSA conjugates to generate the fluorescence on the test line. Negligible nonspecific adsorption was detected for all of the other samples. Consequently, the fluorescence ratio of T/C of the sample containing PSA was much higher than that of other samples (Figure 2B). This indicates that the Pdot-based ICTS possesses the very high specificity towards the target analytes with minimal false-negative or false-positive readout. For practical use of this Pdot-based ICTS in POC diagnostics, the rapid result determination is of importance. In this work, 100 L of samples containing 5 ng/mL PSA was added into the sample pad and the fluorescence ratios of T/C were monitored over time. As shown in the inset of Figure 2B, a detectable T/C ratio appeared after 1 min of sample addition. The T/C ratio reached a plateau at 10 min, indicating that 10 min of reaction time is enough for the accurate readout in this ICTS platform. Quantitative Detection of Cancer Biomarkers in Simulated Physiological Samples. To access the detection sensitivity of the Pdot-based ICTS, we prepared simulated physiological samples by spiking known amount (0-20 ng/mL) of tumor markers (PSA/AFP/CEA), 4 L serum, and 40 L 5% (w/w) PEG400 in 40-60 L 20 mM HEPES buffer. The addition of serum helped simulate the real samples and at the same time greatly reduced the non-specific adsorption of Pdots on the reaction membrane. The PEG400 could effectively aid the migration of Pdots from the conjugate pad to membrane without sticking on the conjugate pad. The detection results were revealed in Figure 3, in which the emission brightness of the test lines increased gradually with the increasing concentrations of tumor markers, accompanied by the decreasing fluorescence in the control lines. This phenomenon can be ascribed to the fact that the total amount of antibodyPdot conjugates is constant on the conjugate pad for each test strip. The higher number of Pdots anchored onto the test zone in the presence of antigens would result in the decreased number of Pdots bound to the control zone. It is worth mentioning that we optimized the concentration of detection/capture antibodies and the amount of Pdot-antibody conjugates in an effort to create a turning point that could be used to quickly decide if the tumor marker level is suspicious or not. By taking PSA as an example (Figure 3A), PSA levels of 4 ng/mL or lower are considered as normal in most cases. In the Pdot-based ICTS, we could clearly observe that the fluorescence intensity of the test zone was lower or at most equal to that of the control zone for PSA levels of 0-3 ng/mL. Once the PSA levels were greater than 5 ng/mL, the

A PSA

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Antigen Figure 2. Performance of the specificity of the PF-TC6FQ Pdotbased ICTS platform, in which the test line was fabricated with PSA capture antibody and the control line was fabricated with bare IgG. (A) Images of the test strips under 365 nm light irradiation after reaction with samples containing different antigens (PSA, AFP, CEA at 5ng/mL), FBS, and RPMI-1640 medium. (B) Their corresponding fluorescence ratios of T/C measured by ImageJ software. The inset in (B) shows the T/C fluorescence ratios at different reaction time.

Mechanism of the Pdot-Based ICTS. After the successful decoration of detection antibody on the surface of Pdots, we would like to integrate them with the test strips for the detection of cancer biomarkers. As illustrated in Scheme 1, the reaction membrane was modified with capture antibody and bare IgG on the test zone and the control zone, respectively. The conjugate pad was modified with Pdot-antibody conjugates. Once the samples were dropped onto the sample pad, the liquid migrated through the test strip by capillary force. In the presence of target analytes (PSA/AFP/CEA), Pdot-antibody conjugates would be captured onto the test line specifically via sandwich immunoreaction, generating a


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Figure 3. Quantitative detection of cancer biomarkers by Pdot-based ICTS platforms. (A) Images of the test strips under 365 nm light irradiation after reaction with samples containg (A) PSA, (B) AFP, and (C) CEA antigens (0-20 ng/mL). The bottom panels show their corresponding fluorescence ratios of T/C measured by ImageJ software. The insets in each panel show the calibration curves at antigen concentrations of 3-15 ng/mL. Error bars show standard deviations of 5 replicate measurements.

fluorescence intensity of the test zone became higher than that of the control zone, of which levels the doctors would recommend a biopsy to further confirm whether cancer was present or not. The same phenomena could also be observed for AFP (>10 ng/mL) and CEA (>5 ng/mL) as shown in Figure 3B,C. These results demonstrated that the Pdot-based ICTS can be used for on-the-spot quick and high-throughput quantitative screening of cancer biomarkers in clinic. We further assessed the detection limit of the Pdot-based ICTS for PSA, AFP and CEA. As shown in the insets of Figure 3, the linear relationship between the fluorescence ratios of T/C and log[PSA/AFP/CEA] concentrations was displayed within the range of 3-15 ng/mL. The limit of detection was estimated by 3/m, where  is the standard deviation of the blank and m is the slope in the linear range. The limit of detection of the Pdot-based ICTS was calculated to be 2.05, 3.30, and 4.92 pg/mL for PSA, AFP, and CEA, respectively.

A PFO PFCN PFTC6FQ

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1.00 ± 0.13 5.49 ± 0.72 15.6 ± 0.94 1.02 ± 0.08 5.33 ± 0.45 15.4 ± 1.31 1.06 ± 0.10 5.27 ± 0.53 15.9 ± 1.52

100 109 104 102 107 103 106 105 106

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CEA (+) AFP (+) PSA (+)

Figure 4. (A) Schematic showing the fabrication of multiplexed ICTS for simultaneous detection of multiple cancer biomarkers. (B) Photographs of detection results of test strips under different antigen concentrations. CEA/AFP/PSA concentrations were as follows (from left to right): 0/0/5, 0/5/0, 5/0/0, 5/5/5 ng/mL.

Table 1. Precision of the Pdot-Based ICTS in Antigen-Spiked Samples. [PSA] nominal (ng/mL)

CEA (+) AFP (−) PSA (−)

PFO-CEA PFCN-AFP TC6FQ-PSA

Determination of PSA in Real Samples. To further demonstrate the practical applications of this platform in real samples, we spiked known amount of antigens into normal human serum samples and then tested the samples (N = 5) by Pdot-based ICTS. As summarized in Table 1, the results show excellent agreement between the experimentally determined values and the spiked concentrations. The recoveries were all around 100% with CV ranging from 6.0 to 13.1 %, indicating the feasibility of the use this Pdot-based ICTS platform for tumor marker detection in clinical samples.

sample

CEA (−) AFP (+) PSA (−)

Multiplexed Detection of Tumor Markers. The multiplexing capability of Pdots are attributed to their broad excitation and distinct emission spectra. To realize the simultaneous detection of tumor markers by Pdot-based ICTS, three corresponding capture antibodies were immobilized separately onto the same test strip as shown in Figure 4A. Three conjugate pads consisting of three different Pdot-antibody conjugates (PFTC6FQ-PSA, PFCN-AFP, and PFO-CEA) were stacked on top of each other and then all of

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them were attached onto the nitrocellulose membrane. The cross

reactivity of the multiplex-capable ICTS was first evaluated as shown in Figure 4B. We could clearly observe that once a single tumor marker was present in the analyte samples, only the corresponding test line exhibited a positive fluorescent signal while other test lines displayed negligible fluorescence. This indicates that each of the three antibodies were specific to their corresponding antigens with minimal cross interference. We further examined the feasibility of the multiplexing ability of this ICTS by loading three tumor markers (PSA, AFP, and CEA) all together into the analyte samples. As shown in the right panel of Figure 4B, all of the three test lines displayed distinct RGB fluorescence from Pdots although inevitable non-specific adsorption of trace Pdots on the nitrocellulose membrane made its background slightly brighter. These results demonstrated that this multicomponent Pdot-based ICTS holds great promise for developing new generations of POC techniques in clinical applications.

CONCLUSIONS In summary, we have developed a sensitive, selective, rapid, and efficient Pdot-based ICTS platform for quantitative detection of cancer biomarkers. By taking advantages of the ultrahigh brightness of the Pdot-based assays in the ICTS platform, we could sensitively and specifically determine the concentrations of PSA/AFP/CEA antigens by using < 60 L serum samples in 10 min with sensitivity of more than 2 orders of magnitude lower than that of the conventional fluorescence-based ICTS.4,46 More importantly, we have successfully realized multiplexed detection of tumor markers by using this platform. We anticipate this Pdotbased ICTS to find broad use for early diagnosis or regular monitoring of cancers or other diseases at the site of patient care.

AUTHOR INFORMATION Supporting Information Synthetic schemes of polymers and complete image of gel electrophoresis. This information is available free of charge via the Internet at http://pubs.acs.org.

Corresponding Author *E-mail: [email protected]

Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT We would like to thank the Ministry of Science and Technology (105-2113-M-110-012-MY3), NSYSU-KMU Joint Research Project (106-P017), and National Sun Yat-sen University.

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Multiplexed Detection of Tumor Markers with Multicolor Polymer Dot

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