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Biological and Medical Applications of Materials and Interfaces
Colorimetric and Fluorescent Dual-Mode Immunoassay Based on Plasmon Enhanced Fluorescence of Polymer Dots for Detection of PSA in Whole-Blood Pei-Yun You, Fang-Chu Li, Ming-Ho Liu, and Yang-Hsiang Chan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b00204 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 23, 2019
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ACS Applied Materials & Interfaces
Colorimetric and Fluorescent Dual-Mode Immunoassay Based on Plasmon Enhanced Fluorescence of Polymer Dots for Detection of PSA in Whole-Blood Pei-Yun You,a Fang-Chu Li,a Ming-Ho Liu,a and Yang-Hsiang Chan*ab a
Department of Applied Chemistry, National Chiao Tung University, Hsinchu, Taiwan 30050 Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan 80708
b
ABSTRACT: Although enormous efforts have been devoted to the development of new types of fluorometric immunochromatographic test strip (ICTS) with improved sensitivity over the past years, it still remains a big challenge to design ICTS with colorimetric and fluorescent bimodal signal readout for rapid yet accurate detection of cancer markers in clinic. Scientists have tried to prepare bimodal reporters by combining fluorescent dyes with metal nanomaterials but their fluorescence was easily quenched by metal nanomaterials through surface energy transfer, making dual colorimetric and fluorometric ICTS very difficult to be achieved. As compared to conventional fluorescent probes, semiconducting polymer dots (Pdots) exhibit extraordinary fluorescence brightness and facile surface functionalization which are very suitable to be engineered as bimodal signal reporting reagents. Here we integrated highly fluorescent Pdots with strongly plasmonic Au nanorods to form Pdot-Au hybrid nanocomposites with dual colorimetric and fluorescent readout abilities. We further utilized these nanohybrids in ICTS for qualitatively fast screening (colorimetry) as well as quantitatively accurate determination (fluorometry) of prostate-specific antigen (PSA) within 10 min. By taking advantage of the plasmon enhanced fluorescence of Pdots on Au nanorods, this immunoassay possesses much better detection sensitivity of 1.07 pg/mL for PSA, which is at least 2 orders of magnitude lower than that of conventional fluorometric ICTS. Moreover, the direct detection of PSA from human whole blood collected without sample pretreatment makes this Pdot-based ICTS platform promising for on-site point-of-care diagnostics. Keywords: semiconducting polymer dots, dual-mode test strip, biomarker detection, PSA POC monitoring, whole-blood analysis
INTRODUCTION
distinguished simply from the shades of signal generating reagents. As a result, enormous efforts have devoted to the development of new reporting reagents in an effort to enhance the detection limit of ICTS.12-14 These new types of reporting reagents usually possess fluorescent properties, which include small organic fluorophores, carbon-based nanodots, upconverting nanoparticles, inorganic quantum dots, and dyeembedded nanomaterials.4, 15-21 The main reason to adopt fluorescent labels in ICTS is because fluorometric ICTS has much better detection sensitivity, higher contrast, and lower background interference as compared to traditional colorimetry-based ICTS, allowing for the quantitative detection of trace amount of targets.22 In our previous work, we have successfully employed multicolor semiconducting polymer dots (Pdots) as fluorescent probes in ICTS to simultaneously detect different types of cancer markers.13 The Pdot-based ICTS exhibited excellent detection sensitivity of at least 2 orders of magnitude lower than that of present fluorometric ICTS, owing to the extraordinarily high fluorescence brightness, large absorption cross-sections, and large Stokes shifts of Pdots.23-37 Although Pdot-based ICTS appears to be superior to other fluorometric ICTS in terms of detection sensitivity, there still exist several challenges that might impede its practical usage in clinic. First, the purchase of a professional lateral flow test strip reader for quantitative fluorescence measurement might be a burden for patients. Furthermore, even many fluorescence readers can be made portable, it would be much more straightforward if the test results can be directly viewed or analyzed by a smartphone or laptop. The above-mentioned limitations of current
Point-of-care (POC) diagnostics is becoming a significant clinical-medicine subject which offers the possibility of providing rapid diagnostic results in a nonlaboratory environment, making it particularly advantageous for on-thespot patient care.1-2 Additionally, POC test enables a one-step, fast-response, easy-to-operate, and cost-effective methodology for detecting target(s) of interest in complex media, which is very suitable for preliminary disease diagnosis and instant clinical-medical treatment. Lateral flow-based immunoassay or immunochromatographic test strip (ICTS) or is first developed by Unipath3 in late 1900s and has become the most commonly used diagnostic manner of POC in clinical diagnosis, drug-of-abuse testing, food safety, therapeutic monitoring, healthcare analysis, environmental detection, and law enforcement.1-2, 4-11 Most of the commercially available ICTS platforms are based on colloidal gold nanoparticles2 due to their strong absorbance in the visible region to generate prominent colors. While the signal reporting reagents have significant impact on the detection sensitivity, the selection of an appropriate reagent is of importance to fabricate ICTS with high accuracy and reliability. To date, most gold nanoparticle-based ICTS employs the colorimetric method in which the distinct colors from gold nanoparticles (usually red) on the test strips can be promptly visualized by naked eyes. The colorimetry-based ICTS is great for qualitative or semiquantitative detection but still remains a challenge for more precise quantitative readout because the subtle changes of analyte concentrations cannot be
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fluorometry-based ICTS motivated our group to design a new type of ICTS platform with dual colorimetric and fluorescent readout capabilities. The colorimetric method allows for prompt identification without the need of professional operation. The fluorescent signals, on the other hand, can be used for more accurate quantitative analysis to determine if a further precise medical diagnosis and intervention is required for subjects.
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membranes (pore size = 8 m, CNPC), sample pads (GFB-R4), conjugate pads (PT-R5), and absorbent materials (AP080) were obtained from Advanced Microdevices Private Limited (mdi) and modified to 1.5-2 mm in width for assembly into test strips and plastic cassettes. Preparation of Au Nanorods. Au nanorods were prepared by the two-step method according to the previously reported work with modified procedures.46 Briefly, the Au seed solution was first synthesized by adding 15.15 L of pure water, 9.85 L of HAuCl4 (10 mg/mL), and 1 mL of cetyltrimethylammonium bromide (CTAB, 0.1 M) into a 1.7 mL Eppendorf tube sequentially. After the gentle mixing, a freshly prepared 55 L of NaBH4 (0.01 M) was quickly injected into the mixed solution and then vigorously shaken for 2 min. The seed solution was kept still at 30 °C in a water bath for 2 h before further use. For the growth of Au nanorods with the absorption maximum at 650 nm (Au650), 303 L of pure water, 197 L of HAuCl4 (10 mg/mL), 10 mL of 0.1 M CTAB, 45 L of 0.01 M AgNO3, and 200 L of 1 M HCl were added into a 15 mL centrifuge tube and then mixed well. After that, 80 L of 0.1M ascorbic acid was added and the color of the solution turned from yellow to transparent. Finally, 24 L of the seed solution was added and left undisturbed at 30 °C in a water bath for 6 h, followed by the addition of 200 L of 1 M NaOH. For the preparation of Au550 or Au800 nanorods, the amount of AgNO3 was changed to 20 L and 60 L, respectively. The absorption spectra of the resulting Au nanorods were characterized by UV-visible spectroscopy (Dynamica Halo DB20S, Dynamica Scientific).
In this study, we report the bimodal readout ICTS based on Pdots for the colorimetric and fluorescent detection of prostate-specific antigen (PSA), a tumor marker of prostate cancer which has been widely studied.38-41 Specifically, we coated the surface of gold nanorods with Pdots to serve as the reporting reagent. In this scenario, the Pdot-capped Au nanorods possessed both surface plasmon resonance and fluorescent properties, rendering this ICTS platform feasible for bimodal readout. Moreover, the fluorescence of Pdots can also be enhanced by the electromagnetic coupling with Au surface plasmons,42-45 which is beneficial to achieve better detection limit. On the test strip, we modified the capture on the control line and modified by IgG antibody on the test line for the qualitative detection and quantitative determination of PSA from the color contrast and the fluorescence ratio of the test line to control line, respectively. We further employed this bimodal ICTS platform for rapid determination of PSA tumor marker in human whole blood samples. More importantly, only trace blood volume (
