Enhanced Fluorescence ELISA Based on HAT ... - ACS Publications

Subscriber access provided by University of Newcastle, Australia

Article

Enhanced Fluorescence ELISA Based on HAT Triggering Fluorescence “turn-on” with Enzyme-antibody Dual Labeled AuNP Probes for Ultrasensitive Detection of AFP and HBsAg Yudong Wu, Weisheng Guo, Weipan Peng, Qian Zhao, Jiafang Piao, Bo Zhang, Xiaoli Wu, Hanjie Wang, Xiaoqun Gong, and Jin Chang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16236 • Publication Date (Web): 02 Mar 2017 Downloaded from http://pubs.acs.org on March 7, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Enhanced Fluorescence ELISA Based on HAT Triggering Fluorescence “turn-on” with Enzyme-antibody Dual Labeled AuNP Probes for Ultrasensitive Detection of AFP and HBsAg Yudong Wu a, Weisheng Guob, Weipan Peng a, Qian Zhao a, Jiafang Piao a, Bo Zhang a, Xiaoli Wu a

a

, Hanjie Wang a, Xiaoqun Gong *a and Jin Chang *a

School of Materials Science and Engineering, School of Life Sciences, Tianjin University and

Tianjin Engineering Center of Micro-Nano Biomaterials and Detection-Treatment Technology (Tianjin), 92 Weijin Road, Nankai District, Tianjin 300072, P. R. China b

CAS Key Laboratory for Biological Effects of Nanomaterials & Nanosafety National Center for

Nanoscience and Technology, Beijing , 100190 , China

E-mail address: Xiaoqun Gong, [email protected] Jin Chang, [email protected]

ABSTRACT: At present, enzyme-linked immunosorbent assay (ELISA) was considered to be a kind of the most appropriate approaches in clinical biomarker detection, with good specificity, low cost, and straightforward readout. However, the unsatisfactory sensitivity severely hampered its wide application in clinical diagnosis. Herein, we designed a new kind of enhanced fluorescence enzyme-linked immunosorbent assay (FELISA) based on the human alpha-thrombin (HAT) triggering fluorescence “turn-on” signals. In this system, detection antibodies (Ab2) and HAT were labeled on the gold nanoparticles (AuNPs) to form the detection probes, and a bisamide derivative of Rhodamine110 with fluorescence quenched served as the substrate of HAT. After the sandwich immunoreaction, HAT on the sandwich structure could catalyze the cleavage of the fluorescence-quenched substrate, leading to strong fluorescence signal for sensing ultralow levels of alpha fetoprotein (AFP) and hepatitis B virus surface antigen (HBsAg). Under the optimized reaction conditions, AFP and HBsAg were detected at the ultralow concentrations of 10-8 ng mL-1 and 5 × 10-4 IU mL-1, respectively, which were at least 104 times lower than that of the conventional fluorescence assay and 106 times lower than that of the conventional ELISA. In addition, we further discussed the efficiency of the sensitive FELISA in clinical serum samples, showing great potential in practical applications.

Keywords: fluorescence enzyme-linked immunosorbent assay, gold nanoparticles, human alpha-thrombin, alpha fetoprotein, hepatitis B virus surface antigen

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 18

INTRODUCTION: Recently, detection of biomarkers with ultrahigh sensitivity gained intense attention due to its enormous function in early stage disease diagnosis, biomarker content monitoring, and therapeutic information feedback1-4. It has been reported that early diagnosis may help to improve therapeutic intervention5. As known to all, ELISA was the most popular immunoassay in clinical biomarkers detection, due to its good specificity, low cost, and straightforward readouts6-7. However, the sensitivity of conventional ELISA was powerless to screen ultralow concentration of biomarkers in some diseases early stages8-9. Thus, there was an urgent need to develop ultrasensitive detection methods for kinds of biomarkers. At present, fluorescence immunoassay, showed good compatibility with the currently available analytical platforms, was considered to be one of the most sensitive approaches for screening biomarkers10-14. Hence, the marriage of ELISA and fluorescence immunoassay showed exciting clinical diagnosis potential due to the complementary diagnostic capacity, known as FELISA. In the past few decades, many efforts focused on the promising FELISA, including horse radish peroxidase (HRP)-based chemiluminescent immunoassays15, alkaline phosphatase (ALP)-based

chemiluminescent

immunoassays16,

enhanced

luminescence

enzyme

immunoassay17-19, and so on. However, the fundamental limitations of fluorescence immunoassays, such as serious fluorescence quenching and an unsatisfactory sensitivity, severely hampered its application in clinical diagnosis. In other words, the sensitivity of these conventional fluorescence methods still cannot satisfy the ultrasensitive detection requirements. And now one of the most effective approaches to improve the sensitivity of FELISA was searching for a highly efficient enzyme and its corresponding high performance substrate. HAT, a kind of highly specific serine protease, showed a strong catalytic performance in cleaving corresponding peptide at the Arg site20. Based on the advantages of HAT, researchers designed kinds of bisamide Rhodamine110 substrate for the detection of HAT in serum with a high sensitivity21. These bisamide Rhodamine110 substrates with fluorescence quenched, mainly obtained from Rhodamine110’s amino groups linked to peptide. After the enzymatic hydrolysis, the fluorescence-quenched bisamide Rhodamine110 substrates were cut into weak fluorescent monoamide Rhodamine110 and then into the strong fluorescent Rhodamine110, with a further increase in fluorescence signal intensity, which was called “graded enzymatic hydrolysis”

22-23

.

And if the HAT was introduced as the labels in FELISA, it may help to solve the fundamental limitations of fluorescence immunoassay effectively. In this way, the detection sensitivity will certainly be improved. Herein, we introduced an enzyme-antibody dual labeled AuNP probe multi-HAT-AuNP-Ab2 into an ELISA signal generation system to realize the ultrasensitive detection. It is generally


Page 3 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


known that AuNPs possessed an ability to immobilize kinds of molecules onto AuNPs24-28. In this work, detection antibodies (Ab2) and HAT were labeled on the gold nanoparticles (AuNPs) to form the detection probes, and a bisamide derivative of Rhodamine110 with fluorescence quenched served as the substrate of HAT. As shown in Scheme 1, HAT was chosen, instead of commonly used HRP, as the model enzyme to prepare the immune fluorescence probe for the detection of AFP and HBsAg. After the sandwich immunoreaction, HAT on the sandwich structure could catalyze the cleavage of the fluorescence-quenched substrate, triggering strong fluorescence signal “turn-on”. In addition, this fluorescence immunoassay was also used to detect patient serum samples for investigating the efficiency and robustness. The signal enhancement was realized mainly by three factors. One was based on the high catalytic activity of HAT to generate strong fluorescence signal. Another factor was based on the largely proteins loading of AuNPs to further amplify the fluorescence signals. The third one was based on the low background signal due to the so-called “graded enzymatic hydrolysis” of the nonfluorescent Rhodamine110 bisamide substrate.

Scheme 1. Schematic diagram of the enhanced FELISA for detection of AFP and HBsAg. (a) Procedures of the proposed immunoassays with the newly dual labeled AuNP probes mluti-HAT-AuNP-Ab2; (b) Enzymatic hydrolysis of the bisamide Rhodamine110 substrates.

EXPERIMENTAL SECTION: Materials and Instrumentation. The bisamide substrate of HAT, bis (p-tosyl-Gly-Pro-Arg) derivative of Rhodamine110, was purchased from Thermo Fisher Scientific. HAT was supplied by Haematologic Technologies Inc. (Essex Junction, VT). Tetrachloroauric acid trihydrate (HAuCl4), glutaraldehyde, trisodium citrate were supplied by Sigma. Mouse monoclonal primary anti-human AFP antibody, secondary anti-human AFP antibody, AFP antigen, Anti-HBsAg antibody and HBsAg were purchased from Bioscience Diagnostic Technology Co. (Tianjin, China). The 96 well polystyrene plates were obtained from Corning Costar. Tween-20 was purchased from Alfa Aesar. Bovine serum albumin (BSA), fetal bovine serum (FBS), lysine hydrochloride, and polyethylene glycol (PEG,



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

number-average molecular weight (Mn) = 20000) were supplied by DG Biotechnology Co. (Beijing, China). Sodium periodate, ammonium chloride, sodium boron hydride, potassium carbonate, nitric acid and hydrochloric acid was supplied by JT Chemical Reagent (Tianjin, China). The serum samples were collected from Tianjin Medical University General Hospital. Deionized water used in this work has a resistivity of 18.2 MΩ· cm, which was Milli-Q grade. The ultraviolet-visible spectrum of AuNPs were measured by an ultraviolet-visible spectrophotometer (PerkinElmer, United States). TEM images (size and morphology) of all AuNPs were recorded with analytical electron microscope. We recorded the Dynamic light scattering (DLS) with a Zeta Potential Analyzer (BrookHaven Instruments Corporation). The fluorescence intensity in 96 well polystyrene plate was detected by an EnSpire Multilabel Reader (PerkinElmer, United States) at 521 nm. The fluorescence images of Rhodamine110 was monitored with a signal acquisition device-Azure C600 (Azure Biosystems, Inc).

Synthesis and Characterization of AuNPs. All glass service were immersed in aqua regia, washed three time with deionized water, and stored at 150 °C. AuNPs were prepared according to the previous reports29-31. To prepare smaller-sized AuNPs (< 30 nm), 100 mL of HAuCl4 solution (0.01 %) was heated to boiling, 2.5 or 1.8 mL of 1% trisodium citrate solution was injected into the boiled HAuCl4 solution for 10 min to obtain AuNPs (21.0 ± 1.9 nm and 26.4 ± 2.5 nm). To prepare larger-sized AuNPs (30 - 70 nm), 150 mL of trisodium citrate (2.2 mM) in deionized water was heated to boiling, 1mL of HAuCl4 (25 mM) was injected into the boiled trisodium citrate for 10 min to obtain a soft pink monodisperse suspension as Au seeds (16.7 ± 0.9 nm). As soon as the Au seeds was prepared, the system was cooled down to 90 °C, and 1 mL of HAuCl4 solution (25 mM) was injected at 90 °C for 30 min to obtain larger-sized AuNPs (35.5 ± 2.6 nm).This process was repeated twice to increase AuNP size (41.2 ± 2.7 nm). After that, 55 mL of the above solution was added into 2 mL of 60 mM sodium citrate and 53 mL deionized water as a seed solution to prepare AuNPs with larger sizes by the same method (48.5 ± 4.1 nm, 54.4 ± 5.6 nm, and 64.7 ± 5.7 nm).

Preparation of Enzyme-antibody Dual Labeled AuNP Probes. Multi-HAT-AuNP-Ab2 conjugates were prepared as follows32-34. Firstly, the pH value of the AuNPs solution went up to 8.0-8.5 with a solution of potassium carbonate (0.1 mol L-1). The mixture solution of Ab2 (1 mg mL-1) and HAT (1 mg mL-1) with a ratio of 1:1-5 was added into above AuNPs solution. After that, the mixture was agitated for 20 min and stood for 120 min. 100 µL of polyethylene glycol (PEG, 1.0 %) was added to the result solution, and incubated for 30 min. Multi-HAT-AuNP-Ab2 conjugates were centrifuged for 30 min with a centrifugal force of 10, 100


Page 4 of 18

Page 5 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


g and stored at 4 °C in PBS buffer (1 mg mL-1 sodium azide and 10 mg mL-1 BSA).

Procedures of Enhanced FELISA We performed the the enhanced FELISA in 96 well polystyrene plate as follows10,24: The 96 well polystyrene plates were incubated with 100 µL of capture antibodies (0.01 mg mL-1) at 4 °C for 12h. Unbound capature antibodies were removed from the plates with washing buffer (1 % PBST), and then the plates were incubated with 300 µL blocking reagent (0.1 % BSA) over a period of 60 min at 37 °C. After that, the plate was washed three times with washing buffer, 100 µL of analyte standards were added into each well of the plates with various concentrations, and incubated over a period of 60 min at 37 °C. Subsequently, the plate was washed three times with washing buffer, and 100 µL of the dual labeled multi-HAT-AuNP-Ab2 probes were added and incubated over a period of 30 min at 37 °C to form the sandwiched immunocomplex. And then, the microplate was washed five times with 1 % PBST and one times with deionized water, to wipe off redundant HAT. Finally, 2 µL of the bisamide substrate (1.59 mM in 10 mM PBS, pH 7.4) and 98 buffer was added into each well of the plates for the enzyme reaction. Then the solution in each well of the plates was used for the fluorescence detection with a 498 nm-excitation and a 521 nm-emission.

Preparation of HAT-Ab2 Conjugates. HAT-Ab2 conjugates were prepared by a classical glutaraldehyde (GA) crosslinking method35. GA was a commonly used bifunctional crosslinking agent, two aldehyde groups of GA will connect with a free amino group or phenolic group of proteins to form covalent bond. First, 1 mg of HAT was dispersed in a 1.25 % glutaraldehyde solution (0.1mol L-1 PBS, pH 6.8) to form HAT-GA conjugates. The conjugates were purified by dialysis to remove excess GA. And then, 0.5 mg of Ab2 in carbonate buffer (1 mol L-1, pH 9.5) was injected into the above solution, and incubated overnight at 4 °C to form HAT-GA-Ab2 probes. After that, the solution was incubated with lysine hydrochloride to block HAT-GA-Ab2, and then purified by dialysis (0.15 mol L-1 PBS, pH 7.4) again. Resulting solution was purified by centrifugation to remove impurity substance, and stored at 4°C. The HRP-Ab2 probes was prepared by a sodium periodate method, which was similar to the preparation process of HAT-Ab2 conjugates36.

Preparation of FITC-Ab2 Conjugates. The ITC (-N=C=S) group of FITC could connect with the amino-group of Ab237. Firstly, the pH of Ab2 solution was adjusted to 9.0 by dialysis (0.1mol L-1 carbonate buffer). And then, 150 µL of freshly prepared FITC solution (1 mg mL-1, DMSO) was injected into the Ab2 solution.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

After that, the solution was shaken at room temperature for 8 h in the dark, and a solution of ammonium chloride was added into the system to terminate the reaction with a final concentration at 0.05 mol L-1. Finally, the unreacted FITC was removed by dialysis (0.15 mol L-1 PBS, pH 7.4), and the final product was stored at 4°C.

Detection of the Clinical Samples Five serum samples from patients who suffered from liver disease were obtained from Tianjin Medical University General Hospital. The detection of the clinical samples was approved by Tianjin Medical University General Hospital. For AFP detection, we diluted five patient serum samples 10, 1000, 105, 107 and 109 times using PBS, and detected by enzyme-labeled HAT-Ab2 and our multi-HAT-AuNP-Ab2 dual labeled probe. For HBsAg detection, we diluted six HBsAg standard samples 10, 100 and 1000 times using FBS, and detected by our multi-HAT-AuNP-Ab2 dual labeled probe.

RESULTS AND DISCUSSION: Protocol of the Enhanced FELISA In this work, a new kind of FELISA was successfully constructed based on the high catalytic activity of HAT triggering fluorescence “turn-on” of the substrate for the first time. As we can see from the Scheme 1, the detection was performed on PS plate, capture antibodies were immobilized onto the PS plate. In the presence of targeted biomarker, dual labeled AuNP probe multi-HAT-AuNP-Ab2 will be captured to form an immune sandwich structure. The HAT molecules on the immune sandwich structure could catalyze the bisamide Rhodamine110 substrate through the cleavage of the substrate at the Arg site. Upon graded enzymatic hydrolysis, fluorescence quenched bisamide Rhodamine110 substrates were cut into weak fluorescent monoamide Rhodamine110 and then into the strong fluorescent Rhodamine110, which contributed to a lower background signal (Figure 1a, 1b)

22-23

. A solution of the bisamide Rhodamine110

substrate was used to confirm that HAT has been immobilized onto the surface of AuNPs. We investigated the effect of the several assay components on the substrate cleavage and ensured that the cleavage of the substrate was only caused by HAT or multi-HAT-AuNP-Ab2 probes and not by any other side reaction or component present in the assay, which suggested HAT was successfully immobilized onto the surfaces of AuNPs (Figure 1d, 1e). And the immobilization of Ab2 was also confirmed by a classical gold immunochromatographic test, which suggested that the multi-HAT-AuNP-Ab2 probes have excellent immune activity (Supporting Information Figure S2).


Page 6 of 18

Page 7 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Characterization of the FELISA test. (a) Constitutional formula of bisamide substrate, monoamide substrate and Rhodamine110, and the bisamide substrate was non-fluorescent, the monoamide substrate was weak fluorescent, while Rhodamine110 was strong fluorescent; (b) Diagram of the non-fluorescent intramolecularly quenched bisamide substrate and its fluorescent cleavage products; (c) Calibration curves of HAT at concentration of 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8, 3.9, 1.95 ng mL−1 diluted in PBS buffer; (d) Fluorescence intensity of the 96-well PS plate in (e); (e) Photograph showing the cleavage of bisamide substrate upon introduction of AuNP probes. The HAT molecules catalytic degradation the subsrate to strong fluorescent Rhodamine110, and without HAT in the system does not cause catalytic degradation.

Synthesis and Characterization of multi-HAT-AuNP-Ab2 Probes. As the proteins carrier, the property of AuNP also had an important influence on the detection of FELISA. Firstly, the particles size of AuNPs were studied. Spherical AuNPs with eight different sizes were synthesized with uniform and monodispersity (Figure 2 a-p Supporting Figure S1 i). Then AuNPs were decorated with detection antibody Ab2 and HAT through the interaction between proteins and AuNPs32. The particle size was obviously increased after modification with HAT and Ab2 (Supporting Information Figure S1 a-h).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. The characterization of AuNPs with eight different particle sizes. TEM showing eight types of AuNPs: 16.7 nm ± 0.9 nm (a), 21.0 nm ± 1.9 nm (c), 26.4 nm ± 2.5 nm (e), 35.5 nm ± 2.6 nm (g), 41.2 nm ± 2.7 nm (i), 48.5 nm ± 4.1 nm (k), 54.4 nm ± 5.6 nm (m), 64.7 ± 5.7 nm (o). The 100 nm scale bar applies to all images. Statistical histograms (b, d, f, h, j, l, n and p) of particle size distribution and particle size through the corresponding TEM images (a, c, e, g, i, k, m and o) to obtain the results as a rough approximation of the mean diameter ± the standard deviation. One hundred particles for statistics were measured for all particle sizes.

The loading rates of HAT and Ab2 maybe have some connection with the size of AuNP, which can significantly affect the assay sensitivity31. And to discover a more accurate tendency in the size-dependent effect, eight different particle size AuNPs nanoprobes were studied in the sandwich immuoreation by HAT triggering the fluorescence signals. Figure 3a showed the detection result of eight different particle size AuNPs nanoprobes. The ∆F value was calculated to evaluate the performance of the probes multi-HAT-AuNP-Ab2 (∆F= F1 - F0, where F1 was the fluorescence intensity of 1 ng mL−1 antigen and F0 was the fluorescence intensity of negative group ) was calculated, and ∆F value reached the highest by the 26.4 nm AuNPs nanoprobes with a satisfactory fluorescence signal and a moderate background signal, while others suffered from a weaker fluorescence signal or a stronger background signal, which was mainly caused by the effect of particle size27. Smaller AuNPs maybe too small to immobilize enough biomolecules effectively, and the large one was easy to precipitate and hard to be washed away from the 96-well polystyrene plate. So the 26.4 nm AuNPs nanoprobes were chosen to be applied in the FELISA. Since the multi-HAT-AuNP-Ab2 probes were prepared by immobilizing Ab2 and HAT


Page 8 of 18

Page 9 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


molecules onto the surface of AuNPs based on electrostatic interactions, ratios of HAT to Ab2 were key factors affecting the performance of this immunoassay system28. As shown in Fig 3b, the ∆F value increased with the ratios of HAT to Ab2 at 0.5:1, 1:1, 2:1 and 3:1, decreased along with the ratios more than 3:1. Because the detection signal comes from HAT molecules, it was easy to know that the fluorescence signal increased with amounts of HAT, but at the same time the background signal increased due to the nonspecific adsorption. Therefore, the ratio of HAT to Ab2 at 3:1 was chosen in this immunoassay. And the coverage of the antibodies on the Au nanoparticle was estimated by the total amount of proteins (HAT and antibody) and the amount of HAT, the concentration of HAT was 3.6258 × 10-4 g L-1 based on the calibration curve of HAT (Figure 1c), total concentration of proteins were 8.9 × 10-4 g L-1 (Supporting Information Figure S5). The molecular weight of Ab2 was 150 KDa (Dalton), and the concentration of Ab2 was 3.5161 × 10-9 M. The concentration of AuNP probes was 6.8524×10-10 mol L-1 based on an empirical formula: c = A450/ε450 (c represents the concentration of AuNP probes, A450 represents absorption at 450 nm and ε450 was an empirical coefficient which relates to size of AuNP). So the antibodies on the AuNPs were estimated at 5.138-39.

Figure 3. Optimization test. (a) Effect of the size of the AuNPs probes corresponding with as-prepared AuNPs with eight types: 16.7 nm ± 0.9 nm, 21.0 nm ± 1.9 nm, 26.4 nm ± 2.5 nm, 35.5 nm ± 2.6 nm, 41.2 nm ± 2.7 nm, 48.5 nm ± 4.1 nm, 54.4 nm ± 5.6 nm, 64.7 nm ± 5.7 nm; (b) Effect of the molecule ratios, the ratios of Ab2 to HAT was 1/0.5, 1/1, 1/2, 1/3, 1/4, and 1/5; (c) The protein mixture (HAT and Ab2) was 0.004, 0.007, 0.01，0.015, 0.02 mg; (d) The concentration of the multi-HAT-AuNPs-Ab2 probes was 1, 2, 3, 4, 5 ng mL-1.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

In general, an appropriate concentration of proteins (HAT and Ab2) will certainly contribute to improve labeling efficiency in the preparation process of the AuNP probes32. However, excess protein molecules was wasteful and uneconomical, and also could lead to the aggregation of AuNPs and cause an increasing background signal. As we can see from Fig 3c, ∆F value increased with 0.004, 0.007, 0.01, 0.015 and 0.02 mg mL-1 proteins. Considering that there was a slender increasing rate of ∆F value after 0.01 mg mL-1, 0.01 mg mL-1 was chosen for further experiments to reduce proteins consumption. As we all know, concentration of the detection probes was another vital parameter in a FELISA system, which greatly affects the detection sensitivity27. Sometimes inadequate detection probes will not have the capacity to capture all the target analytes, which was harmful to fluorescence intensity and the detection accuracy. And no doubt, excessive dose of detection probes mean a high fluorescence background, which was harmful to detection sensitivity and the detection accuracy, was easy to obtain a false positive result. AuNP probes with different concentrations at 1, 2, 3, 4, and 5 ng mL-1 were used for detection of negative group and 1 ng mL−1 antigen in PBS. Figure 3d showed the optimization of multi-HAT-AuNP-Ab2 probes concentration and probes at concentration of 3 mg mL−1 were the best to be used for this fluorescence immunoassay system.

Procedure of Immunoassay for AFP. AFP was one of most commonly used biomarker for cancer diagnosis in cancerous persons who suffered from primary hepatic carcinoma40. So it was important to monitor ultratrace concentrations variation of AFP to improve cure rate, especially in the early stage of disease. In this work, AFP was chosen as the target to be detected by our dual labeled multi-HAT-AuNP-Ab2 probes. We studied the specificity of this immunoassay for AFP by compared with other tumor markers sharing similar constituent with AFP. As we can see from Figure 4(d), the fluorescence signal of the non-target markers were accordant, also agreed with that of PBS-only blank, and were far below fluorescence intensity of AFP.


Page 10 of 18

Page 11 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Conventional fluorescence immunoassay and the enhanced FELISA for detecting AFP in a 96 well polystyrene plate. (a)

Multi-HAT-AuNP-Ab2: Fluorescence photographs of the newly developed fluorescence probe multi-HAT-AuNP-Ab2 for various concentrations (10-8 ng mL-1 to 10 ng mL-1) of AFP, photographs were taken by an automatic exposure. HAT-Ab2: Fluorescence photographs of the conventional used fluorescence probe HAT-Ab2 for various concentrations (10-8 ng mL-1 to 10 ng mL-1) of AFP, photographs were taken by an automatic exposure. FITC-Ab2: Fluorescence photographs of the conventional used fluorescence probe FITC-Ab2 for various concentrations (10-6 ng mL-1 to 1000 ng mL-1) of AFP, photographs were taken by a prolonged exposure. (b) Comparison of the immunoassay data of the three fluorescence probes by ∆F

max

at 521 nm for various concentrations of AFP in PBS

solutions. (c) The linear range of multi-HAT-AuNP-Ab2 probes and HAT-Ab2 probes. ∆F

max

= F - F0 was calculated to evaluate the

performance (F was the fluorescence intensity of AFP and F0 was the fluorescence intensity of PBST at 521nm). (d) Specificity test. The specificity test was completed with 10 ng mL-1 (BSA, CEA, PSA), 50 IU mL-1 (CA153, CA125, HCG), PBS, 1 ng mL-1 (AFP) antigen. Error bars show standard deviations (n = 3).

Then, we further studied the detecting sensitivity of the designed method. The control experiments were set as enzyme-labeled probe HAT-Ab2 and fluorescein-labeled probe FITC-Ab2. Figure 4 showed the results of the three probes, multi-HAT-AuNP-Ab2 probe showed an excellent detection performance than that of the HAT-Ab2 and FITC-Ab2 probe.The detection limit of the multi-HAT-AuNP-Ab2 probe for AFP, which can be seen as the AFP concentration that yielded an obvious fluorescence signal than the negative group, was 10−8 ng mL-1 (Supporting Information Figure S3), exceeded the detection limit of HAT-Ab2 (10-4 ng mL-1) and FITC-Ab2 (0.1 ng mL-1) by more than at least 4 orders of magnitude (Figure 4a, 4b, 4c). And the superiority of our newly dual labeled AuNP probe was also verified by a most commonly used HRP-ELISA method (Supporting Information Figure S4). The sensitivity of most fluorescence immunoassays was mainly determined by the affinity of antibody, the enzyme labeling efficiency, and the background fluorescence signals. The excellent detection performance of the enhanced FELISA than conventional fluorescence probes was explained by three effects: (1) high loading efficiency of HAT and Ab2, many molecules of HAT and Ab2 could be loaded on a single AuNP, which will enhance local concentrations of enzyme on each immune sandwich structure, compared with



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

conventional fluorescence immunoassays; (2) enzyme-based signal amplification, one molecule of HAT could catalyze lots of the bisamide Rhodamine110 substrate through cleavage of the substrate at the Arg site to turn on the strong fluorescence signals; (3) low background signals, the nonfluorescent Rhodamine110 bisamide substrate was only converted to the weak fluorescent monoamide substrate rather than to strong fluorescent Rhodamine110 for the negative control group, which will be contributed to the reduction of the background signal. In addition, we further investigated the efficiency of the sensitive FELISA for AFP detection in clinical serum samples, compared with enzyme-labeled probe HAT-Ab2. We diluted five patient serum samples 10, 1000, 105, 107and 109 times using PBS, and detected by enzyme-labeled probe HAT-Ab2 and our multi-HAT-AuNP-Ab2 dual labeled probe. As shown in Figure 5, the enzyme-labeled probe HAT-Ab2 could detect AFP only after 105-folds dilution (Figure 5b), where the concentrations of AFP were about 0.01 ng mL-1, while the novel dual labeled AuNP probes could detect AFP even after 109-folds dilution (Figure 5a), which indicated that the dual labeled AuNP probes possess the potential applications in clinic.

Figure 5. Clinical test. Clinical test results of the newly developed dual labeled AuNP probes multi-HAT-AuNP-Ab2 (a) and the

conventional fluorescence probes HAT-Ab2 (b) for patient serum samples suffered from liver cancer. Error bars show standard deviations (n = 3).

Procedure of Immunoassay for HBsAg. To test whether the novel FELISA worked with others biomarkers, HBsAg was also introduced as a model analyte. HBsAg was the coat protein of hepatitis B virus (HBV), which was not infectious, but its presence was often accompanied by the presence of HBV, so it was an effective biomarker of HBV41. First, HBsAg was detected by the dual labeled probe multi-HAT-AuNP-Ab2 with HBsAg spiked into PBS, to fit a calibration curves. And then, albumin from bovine serum (BSA), a kind of protein with low nonspecific absorption, was used to the control experiments. As indicated in Figure 6, the detection of HBsAg performed high specific and the detection limit for HBsAg, was 5 × 10−4 IU mL-1 (Figure 6a, 6b). In addition, we also


Page 12 of 18

Page 13 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


investigated the performance of this assay for HBsAg with six HBsAg samples, they were diluted 10, 100, 1000 times using FBS, and detected by our multi-HAT-AuNP-Ab2 dual labeled probe. The novel dual labeled AuNP probes can detect HBsAg even after 1000-folds dilution (Figure 6c), suggested that this novel FELISA detection system can also transfer to other biomarkers ultrasensitive detection.

Figure 6. Detection of HBsAg with the newly developed dual labeled AuNP probe multi-HAT-AuNP-Ab2 in a 96 well polystyrene plate. (a) Fluorescence photographs of the newly developed dual labeled AuNP probes multi-HAT-AuNP-Ab2 for various concentrations

(10-4 ng mL-1 to 1 ng mL-1) of HBsAg, photographs were taken by a prolonged exposure. (b) Blue: Detection performance of the enhanced FELISA for various concentrations of HBsAg in PBS solutions. Red: The detection results of an unrelated protein BSA. ∆F max = F - F0 was calculated to evaluate the performance (F was the fluorescence intensity of AFP and F0 was the fluorescence intensity of PBST at 521nm). (c) Detection results of the newly developed dual labeled AuNP probes multi-HAT-AuNP-Ab2 for HBsAg in FBS with a serially dilution of 10-fold. Error bars show standard deviations (n = 3).

CONCLUSIONS: Herein, we developed a new kind of enhanced FELISA based on HAT triggering fluorescence “turn-on” for ultrasensitive detection of AFP and HBsAg. Given high catalytic activity of HAT, large protein molecules loading capability of AuNPs and low background fluorescence signal of the non-fluorescent bisamide Rhodamine110 substrate, the detection limit of the newly FELISA was largely lower than that of the enzyme-labeled and fluorescein-labeled immunoassays. The detection was performed on an ELISA system, the most commonly used detection method, which was contributed to the application of this approach in the currently diagnostic platforms with remarkable enhancement in sensitivity. This enzyme-triggered method in this study will certainly



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

have great potential for application in other biomedical diagnostics.

Acknowledgements The authors thank Prof. Dingbin Liu (School of Chemistry, Nankai University) for providing access to some equipment used in this study. This study was financially supported by the National Natural Science Foundation of China (51373117, 51303126, 31401578, 31600800), Tianjin Natural Science Foundation (15JCQNJC03100).

Supporting Information Dynamic light scattering (DLS), gold immunochromatographic test strips and UV-visible absorption spectra for different particle size; fluorescence photographs of the fluorescence probe: multi-HAT-AuNP-Ab2 and HAT-Ab2; the detection performance of HRP-Ab2 probe; and the stability and the reproducibility of the AuNP nanoprobes.

REFERENCES (1) De La Rica, R.; Stevens, M. M. Plasmonic ELISA for the Ultrasensitive Detection of Disease Biomarkers with the Naked Eye. Nat. Nanotechnol. 2012, 7 (12), 821–824. (2) Yang, Q.; Gong, X.; Song, T.; Yang, J.; Zhu, S.; Li, Y.; Cui, Y.; Li, Y.; Zhang, B.; Chang, J.; Quantum Dot-Based Immunochromatography Test Strip for Rapid, Quantitative and Sensitive Detection of alpha Fetoprotein. Biosens. Bioelectron. 2011, 30(1), 145–150. (3) Chen R.; Huang, X.; Xu, H.; Xiong, Y.; Li, Y. Plasmonic Enzyme-Linked Immunosorbent Assay Using Nanospherical Brushes As a Catalase Container for Colorimetric Detection of Uultralow Concentrations of Listeria Monocytogenes. ACS Appl. Mater. Interfaces. 2015, 7(51), 28632–28639. (4) Xianyu, Y.; Wang, Z.; Jiang, X. A Plasmonic Nanosensor for Immunoassay via Enzyme-Triggered Click Chemistry. ACS nano. 2014, 8 (12), 12741–12747. (5) Zheng, T.; Pierre-Pierre, N.; Yan, X.; Huo, Q.; Almodovar, A. J. O.; Valerio, F.; Rivera-Ramirez, I.; Griffith, E.; Decker, D. D.; Chen, S. X.; Zhu, N. Gold Nanoparticle-Enabled Blood Test for Early Stage Cancer Detection and Risk Assessment. ACS Appl. Mater. Interfaces. 2015, 7(12), 6819–6827. (6) Rissin, D. M.; Kan, C. W.; Campbell, T. G.; Howes, S. C.; Fournier, D. R.; Song, L. N.; Piech, T.; Patel, P. P.; Chang, L.; Rivnak, A. J.; Ferrell, E. P.; Randall, J. D.; Provuncher, G. K.; Walt, D. R.; Duffy, D. C. Single-Molecule Enzyme-Linked Immunosorbent Assay Detects Serum Proteins at Subfemtomolar Concentrations. Nat. Biotechnol. 2010, 28 (6), 595–599. (7) Liang, J.; Yao, C.; Li, X.; Wu, Z.; Huang, C.; Fu, Q.; Lan, C.; Cao, D.; Tang, Y. Silver Nanoprism Etching-Based Plasmonic ELISA for the High Sensitive Detection of Prostate-Specific Antigen. Biosens. Bioelectron. 2015, 69, 128–134. (8) Wulfkuhle, J. D.; Liotta, L. A.; Petricoin, E. F. Proteomic Applications for the Early Detection of Cancer. Nat. Rev. Cancer. 2003, 3(4), 267–275.


Page 14 of 18

Page 15 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(9) Xia, N.; Wang, X.; Zhou, B.; Wu. Y.; Mao. W.; Liu, L. Electrochemical Detection of Amyloid-β Oligomers Based on the Signal Amplification of a Network of Silver Nanoparticles. ACS Appl. Mater. Interfaces. 2016, 8(30), 19303–19311. (10) Liang, J.; Liu, H.; Huang, C.; Yao, C.; Fu, Q.; Li, X.; Cao, D.; Luo, Z.; Tang, Y. Aggregated Silver Nanoparticles Based Surface-Enhanced Raman Scattering Enzyme-Linked Immunosorbent Assay for Ultrasensitive Detection of Protein Biomarkers and Small Molecules. Anal. Chem. 2015, 87 (11), 5790–5796. (11) Maiolini, E.; Ferri, E.; Pitasi, A. L.; Montoya, A.; Di Giovanni, M.; Errani, E.; Girotti, S. Bisphenol A Determination in Baby Bottles by Chemiluminescence Enzyme-Linked Immunosorbent assay, Lateral flow immunoassay and Liquid Chromatography Tandem Mass Spectrometry. Analyst. 2014, 139 (1), 318–324. (12) Jiang, W.; Wang, Z.; Beier, R. C.; Jiang, H.; Wu, Y.; Shen, J. Simultaneous Determination of 13 Fluoroquinolone and 22 Sulfonamide Residues in Milk by a Dual-Colorimetric Enzyme-Linked Immunosorbent Assay. Anal. Chem. 2013, 85 (4), 1995–1999. (13) Ren, M. L.; Xu, H.; Huang, X.; Kuang, M.; Xiong, Y.; Xu, H.; Xu, Y.; Chen, H.; Wang, A. Immunochromatographic Assay for Ultrasensitive Detection of Aflatoxin B1 in Maize by Highly Luminescent Quantum Dot Beads. ACS Appl. Mater. Interfaces. 2014, 6(16), 14215–14222. (14) Wan, Y.; Qi, P.; Zhang, D.; Wu, J.; Wang, Y. Manganese Oxide Nanowire-mediated Enzyme-Linked Immunosorbent Assay. Biosens. Bioelectron. 2012, 33 (1), 69–74. (15) Zhou, Y.; Zhou, T.; Zhou, R.; Hu, Y. Chemiluminescence Immunoassay for the Rapid and Sensitive Detection of Antibody Against Porcine Parvovirus by Using Horseradish Peroxidase / Detection Antibody-Coated Gold Nanoparticles As Nanoprobes. Luminescence. 2014, 29(4), 338–343. (16) Herman, D. S.; Ranjitkar, P.; Yamaguchi, D.; Grenache, D. G.; Greene, D. N.; Endogenous Alkaline Phosphatase Interference in Cardiac Troponin I and Other sensitive Chemiluminescence Immunoassays that Use Alkaline Phosphatase Activity for Signal Amplification. Clin. Biochem. 2016, 49(15), 1118–1121. (17) Bi, S.; Yan, Y.; Yang, X.; Zhang, S. Gold Nanolabels for New Enhanced Chemiluminescence Immunoassay of Alpha-Fetoprotein Based on Magnetic Beads. CHEM-EUR J. 2009, 15 (18), 4704–4709. (18) Yang, X.; Guo, Y.; Bi, S.; Zhang, S. Ultrasensitive Enhanced Chemiluminescence Enzyme Immunoassay for the Determination of α-Fetoprotein Amplified by Double-Codified Gold Nanoparticles Labels. Biosens. Bioelectron. 2009, 24 (8), 2707–2711. (19) Lu, L.; Wang, M.; Liu, L.; Leung, C. H.; Ma, D. Label-Free Luminescent Switch-on Probe for Ochratoxin A detection Using a G-quadruplex-Selective Iridium (III) Complex. ACS Appl. Mater. Interfaces. 2015, 7(15), 8313–8318. (20) Zhao, Q.; Li, X.; Le, X. C. Aptamer Capturing of Enzymes on Magnetic Beads to Enhance Assay Specificity and Sensitivity. Anal. Chem. 2011, 83 (24), 9234–9236. (21) Pavlov, V.; Shlyahovsky, B.; Willner, I. Fluorescence Detection of DNA by the Catalytic Activation of an Aptamer/Thrombin Complex. J. Am. Chem. Soc. 2005, 127 (18), 6522–6523. (22) Kupcho, K.; Hsiao, K.; Bulleit, B.; Goueli, S. A. A Homogeneous, Nonradioactive High-Throughput Fluorogenic Protein Phosphatase Assay. J. Biomol. Screening. 2004, 9 (3), 223–231. (23) Kupcho, K.; Somberg, R.; Bulleit, B.; Goueli, S. A. A Homogeneous, Nonradioactive



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

High-Throughput Fluorogenic Protein Kinase Assay. Anal. Biochem. 2003, 317 (2), 210–217. (24) Liu, D.; Huang, X.; Wang, Z.; Jin, A.; Sun, X.; Zhu, L.; Wang, F.; Ma, Y.; Niu, G.; Hight Walker, A. R.; Chen, X. Gold Nanoparticle-Based Activatable Probe for Sensing Ultralow Levels of Prostate-Specific Antigen. ACS nano. 2013, 7 (6), 5568–5576. (25) Zhou, L.; Ding, F.; Chen, H.; Ding, W.; Zhang, W.; Chou, S. Y. Enhancement of Immunoassay’s Fluorescence and Detection Sensitivity Using Three-Dimensional Plasmonic Nano-Antenna-Dots Array. Anal. Chem. 2012, 84 (10), 4489–4495. (26) Niikura, K.; Kobayashi, K.; Takeuchi, C.; Fujitani, N.; Takahara, S.; Ninomiya, T.; Hagiwara, K.; Mitomo, H.; Ito, Y.; Osada, Y.; Ijiro, K. Amphiphilic Gold Nanoparticles Displaying Flexible Bifurcated Ligands as a Carrier for siRNA Delivery into the Cell Cytosol. ACS Appl. Mater. Interfaces. 2014, 6(24), 22146–22154. (27) Jia, C.; Zhong, X.; Hua, B.; Liu, M.; Jing, F.; Lou, X.; Yao, S.; Xiang, J.; Jin, Q.; Zhao, J. Nano-ELISA for Highly Sensitive Protein Detection. Biosens. Bioelectron. 2009, 24 (9), 2836–2841. (28) Li, Y.; Zhou, Y.; Meng, X.; Zhang, Y.; Liu, J.; Zhang, Y.; Wang, N.; Hu, P.; Lu, S.; Ren, H.; Liu, Z. Enzyme-Antibody Dual Labeled Gold Nanoparticles Probe for Ultrasensitive Detection of κ-Casein in Bovine Milk Samples. Biosens. Bioelectron. 2014, 61, 241–244. (29) Frens, G. Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions. Nat. Phys. Sci. 1973, 241 (105), 20–22. (30) Bastús, N. G.; Comenge, J.; Puntes, V. Kinetically Controlled Seeded Growth Synthesis of Citrate-stabilized Gold Nanoparticles of Up to 200 nm: Size Focusing Versus Ostwald Ripening. Langmuir. 2011, 27 (17), 11098–11105. (31) Dou, Y.; Guo, Y.; Li, X.; Li, X.; Wang, S.; Wang, L.; Lv, G.; Zhang, X.; Wang, H; Gong, X.; Chang, J. Size-Tuning Ionization to Optimize Gold Nanoparticles for Simultaneous Enhanced CT Imaging and Radiotherapy. ACS nano. 2016, 10 (2), 2536–2548. (32) Shu, J.; Qiu, Z.; Zhou, Q.; Lin, Y.; Lu, M.; Tang, D. Enzymatic Oxydate-Triggered Self-Illuminated Photoelectrochemical Sensing Platform for Portable Immunoassay Using Digital Multimeter. Anal. Chem. 2016, 88 (5), 2958–2966. (33) Zhou, Y.; Pan, F.; Li, Y.; Zhang, Y.; Zhang, J.; Lu, S.; Ren, H.; Liu, Z. Colloidal Gold Probe-Based Immunochromatographic Assay for the Rapid Detection of Brevetoxins in Fishery Product Samples. Biosens. Bioelectron. 2009, 24 (8), 2744–2747. (34) Wu, W.; Li, J.; Pan, D.; Li, J.; Song, S.; Rong, M.; Li, Z.; Gao, J.; Lu, J. Gold Nanoparticle-Based Enzyme-Linked Antibody-Aptamer Sandwich Assay for Detection of Salmonella Typhimurium. ACS Appl. Mater. Interfaces. 2014, 6(19), 16974–16981. (35) McLean, I. W.; NAKANE, P. K. Periodate-Lysine-Paraformaldehyde Fixative a New Fixative for Immunoelectron Microscopy. J. Histochem. Cytochem. 1974, 22 (12), 1077–1083. (36) Wang, H. K.; Tsai, C. H.; Chen, K. H.; Tang, C. T.; Leou, J. S.; Li, P. C.; Tang, Y. L.; Hsieh, H. J.; Wu, H. C.; Cheng, C. M. Cellulose-Based Diagnostic Devices for Diagnosing Serotype-2 Dengue Fever in Human Serum. Adv. Healthcare Mater. 2014, 3 (2), 187–196. (37) Vira, S.; Mekhedov, E.; Humphrey, G.; Blank, P. S. Fluorescent-labeled Antibodies: Balancing Functionality and Degree of Labeling. Anal. Biochem. 2010, 402(2), 146–150. (38) Haiss, W.; Thanh, N. T.; Aveyard, J.; Fernig, D, G. Determination of Size and Concentration of Gold Nanoparticles from UV-vis Spectra. Anal. Chem. 2007, 79 (11), 4215–4221. (39) Ambrosi, A.; Castañeda, M. T.; Killard, A. J.; Smyth, M. R.; Alegret, S.; Merkoci, A.


Page 16 of 18

Page 17 of 18

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Double-Codified Gold Nanolabels for Enhanced Immunoanalysis. Anal. Chem. 2007, 79 (14), 5232–5240. (40) Ludwig, J. A.; Weinstein, J. N. Biomarkers in Cancer Staging, Prognosis and Treatment Selection. Nat. Rev. Cancer. 2005, 5 (11), 845–856. (41) Choi, Y. H.; Lee, G. Y.; Ko, H.; Chang, Y. W.; Kang, M. J.; Pyun, J. C. Development of SPR Biosensor for the Detection of Human Hepatitis B Virus Using Plasma-Treated Parylene-N Film. Biosens. Bioelectron. 2014, 56, 286–294.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Contents Graphic


Page 18 of 18

Enhanced Fluorescence ELISA Based on HAT ... - ACS Publications

Recommend Documents