Effective Bioactivity Retention of Low Concentration Antibodies on

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Biological and Medical Applications of Materials and Interfaces

Effective Bioactivity Retention of Low Concentration Antibodies on HFBImodified Fluorescence ICTS for Sensitive and Rapid Detection of PSA Bo Zhang, Weichen Gao, Jiafang Piao, Yunjie Xiao, Bin Wang, Weipan Peng, Xiaoqun Gong, Zefang Wang, Haitao Yang, and Jin Chang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02945 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018

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ACS Applied Materials & Interfaces

Effective Bioactivity Retention of Low Concentration Antibodies on HFBI-modified Fluorescence ICTS for Sensitive and Rapid Detection of PSA Bo Zhanga,§, Weichen Gao a,§, Jiafang Piao a, Yunjie Xiaoa ,Bin Wanga, Weipan Penga, Xiaoqun Gonga,d*, Zefang Wang a,b,c*, Haitao Yang c and Jin Chang a* a

School of Life Sciences, Tianjin University, Tianjin Engineering Center of Micro-Nano Biomaterials and

Detection-Treatment Technology, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, China b

State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai University, Tianjin 300071, China.

c

Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China.

d

State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, China

§ B.Z. and W.G. have equal contribution to this paper.

Corresponding Author *Jin Chang: Email: [email protected]. Tel: +86-22-27401821 *Zefang Wang: Email: [email protected] *Xiaoqun Gong: Email: [email protected]

Abstract Nowadays,

increasing

analytical

sensitivity

is

still

a

big

challenge

in

constructing

membrane-based fluorescence immunochromatography test strip (FICTS). However, the bioactivity of antibody (Ab) immobilized on the test line (T line) of porous nitrocellulose membrane (PNM), which directly influences the analytical sensitivity, is less studied. In this work, a novel amphiphilic hydrophobin (HFBI) protein was introduced to modify the T line to effectively retain the Abs’ bioactivity. The results indicated that HFBI could self-assemble on the PNM and immobilize the Abs in the “stand-up” orientation. Compared with the conventional FICTS, the HFBI-modified FICTS with only 0.2 mg/mL of monoclonal Abs on T line, enable more accurately quantitative detection and better sensitivity (0.06 ng/mL for prostate specific antigen (PSA)), which is more than 2 orders of magnitude lower than that of the conventional FICTS with the same concentration of monoclonal Abs on T line. Furthermore, the accuracy of this HFBI-modified FICTS was investigated by testing 150 clinical serum samples and the detection results were coincident with those by electrochemiluminescence immunoassay (ECLIA). Our results provide a novel and promising strategy of Ab immobilization on FICTS for near patient and point-of-care application. Keywords: membrane-based fluorescence immunochromatography test strip (FICTS), bioactivity of antibody, amphiphilic hydrophobin (HFBI), prostate specific antigen(PSA), “stand-up” orientation

1

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Introduction The membrane-based immunochromatography test strip has been widely used for near patient and point-of-care diagnostics due to its advantages of fast, convenient, low-cost and single test1-4. However, the conventional gold nanoparticles based strip commonly just provided qualitative (yes or no) or semiquantitative result with poorer detecting sensitivity5,6，and most FICTS hardly perform the sensitive detection as low as the clinical laboratory medicine, such as enzyme-linked immunoassay (ELISA)7 and ECLIA8, but the issues with ELISA and ECLIA mainly lie in the multi-step processing of samples, long diagnostics times, and the overall cost9. Generally, FICTS mainly consists of the PNM, Abs, and fluorescence signal generating reagents10,11. In order to improve the sensitivity, much effort and cost are spent on the signal-generating reagents of FICTS, including 1) utilization of new-type fluorescent probes, such as quantum dots and up-converting nanoparticles12; 2) packaging multiple fluorescent nanoparticles or dyes to form the high fluorescent nanoprobes, such as fluorescent or dye-doped nanospheres containing lots of fluorescers13,14, 3) surface enhanced Raman scattering (SERS)15,16. Though the analytical sensitivity has been improved to some extent, it is still far below the detecting requirement of trace amount of targets. Actually, another key factor that also directly influences the detecting sensitivity is the bioactivity of Abs immobilized on T line of PNM, and has been less studied. In general, Abs are simply deposited on the T line by spraying, easily resulting in random orientation and multilayer superposition with unexposed Fab of Ab and loss of bioactivity17. The ratio of working Abs on the T line is much decreased with serious waste and rising costs. Therefore, new strategies are required to ameliorate the Abs’ immobilization and improve the detection sensitivity of FICTS. Hydrophobin is a type of amphiphilic fungal protein that can self-assemble into a chemically stable protein membrane18,19. A hydrophobic patch composed of grouped surface hydrophobic amino acids endows HFBI with exceptional amphiphilic properties and drives them spontaneous and rapid self-assembly at hydrophobic/hydrophilic interfaces, such as air/water and oil/water interfaces, where they pack into ordered structures and form remarkably strong and elastic films20. Moreover, the resultant HFBI film exhibits strong immobilization capacity to the interested biomolecules onto its modified surface21. For example, HFBI has been applied as enzyme immobilization matrix on platinum electrode to construct amperometric glucose biosensor with significantly improved detection sensitivity22. The self-assemble HFBI was also introduced to form an intact charged film on the polystyrene and glass slides as a biocompatible substrate to improve the outcome of different biomolecules immobilization23. Considering the self-assemble and efficient immobilization performance, we proposed that HFBI could provide an underlying and promising mechanism for immobilizing Abs on the PNM so as to help improve the detection sensitivity of FICTS. To verify our hypothesis, herein, we designed a HFBI-modified FICTS to detect PSA targets, which was recognized as a valuable biomarker for prostate cancer diagnosis24. X-ray photoelectron spectroscopy 2


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(XPS), scanning electron microscope (SEM) and atomic force microscope (AFM) were introduced to characterize the self-assemble performance of HFBI on the PNM and the “stand-up” orientation of immobilized Abs through the HFBI adsorption. Furthermore, we studied the bioactivity of Abs integrated on the HFBI-modified or HFBI-unmodified T line with different concentration. At the optimal condition, the HFBI-modified FICTS displayed higher sensitivity and less usage of Abs compared with the commercial FICTS. To our knowledge, this is the first time to improve the sensitivity of FICTS by perfectly maintaining the biological activity of Abs, indicating the great potential of HFBI to modify and functionalize of other detecting platform surface in disease diagnosis.

Experimental Section Materials 1-Ethyl-3-(3-dimethyllaminopropyl)-carbodiimide hydrochloride (EDC) was purchased from GL Biochem (Shanghai) Ltd. Sucrose was purchased from Aladdin Industrial Corporation. Polyethylene glycol (PEG) 4,000 and Polysorbate 20 were purchased from Alfa Aesar (China) Chemical Co., Ltd. Potassium carbonate, Cadmium oxide (CdO, 99.5%), selenium powder (Se, 99.99%), sulfur (S, 99.98%), zinc oxide (ZnO, 99.9%), poly(tert-butyl acrylate-co-ethyl acrylate-co-methacrylic acid) (ABC triblock copolymer) and 11-mercaptoundecanoic acid (MUA) were purchased from Tianjin Jiangtian Chemical Co., Ltd. Polyvinylpyrrolidone (PVP) 10,000 was purchased from Sigma-Aldrich, Co., Ltd. Bovine serum albumin (BSA) were purchased from Beijing Dingguo Biotechnology Co., Ltd. (China). The tris (hydroxymethyl) aminomethane-HCl (Tris-HCl) buffer, boracic acid buffer and PBS buffer were purchased from Beijing Leagene Biotechnology Co., Ltd. HFBI was produced from Pichia pastoris25. PSA standard sample, monoclonal Ab, goat anti-mouse IgG, reaction membrane, sample pad, conjugate pad and absorbent pad were supplied by Bioscience (Tianjin) Diagnostic Technology Co., Ltd. Patients serum samples were provided by Tianjin Medical University General Hospital. And the above substances were used as provided. Deionized water (Millipore Milli-Q grade) with a resistivity of 18.2 MΩ·cm was used throughout this study. Automatic dispenser-HM3030 was purchased from Shanghai Kinbio Tech Co., Ltd. The test strip reader was designed by our laboratory. Preparation of QD-based Nanoprobes The water-soluble quantum dots beads were prepared according to a previous protocol published by our group13 and characterized by TEM and fluorescence spectrophotometer. Figure S1, S2 and S3 revealed that the QDs beads have a uniform spherical shape with the emission peak wavelength about 620 nm. In order to get the QD-Ab nanoprobes, water-soluble QDs beads, monoclonal Ab, EDC was added in boracic acid buffer (0.01 M, pH 8.0) at a certain molar ratio(QDs/ Ab /EDC=1:16:4000)26. Then the 3



mixture was further incubated for 2 h at room temperature. The QD-Ab nanoprobes were purified by washing and centrifugation. The final QD-Ab nanoprobes were blocked in boracic acid buffer (0.01 M, pH 8.0, 2% BSA) overnight at 4 °C. Fabrication of HFBI-modified FICTS The test strip is mainly composed of a sample pad, conjugate pad, reaction membrane and absorbent pad27-29. The prepared QD-Ab nanoprobes were diluted 10 times in the diluent (pH 7.4, containing 10 mM PBS, 5% (w/v) BSA, 5% (w/v) sucrose, 4%(w/v) PEG4000, and 0.1% (w/v) Tween-20) and a desired volum of QD-Ab nanoprobes were added on the pretreated conjugate pad and dried at 37 ºC for 2 h. 50 µg/mL HFBI was dispensed on T line firstly. Since the adsorption performance of HFBI largely depends on the environmental pH20, K2CO3 was added to adjust the buffer pH as 8.0, 9.0, 10.0 and 10.25, respectively. Then the monoclonal PSA Ab was diluted in the above buffers with the concentration about 1 mg/mL and dispensed on the HFBI-modified T line to get the optimum pH. The goat anti-mouse IgG (1 mg/mL) was dispensed on the C line. Under the optimum pH, the monoclonal PSA Abs were diluted to certain concentrations (0.2, 0.5, 1 and 2 mg/mL) and dispensed on the HFBI-modified T line to verify whether the HFBI can effectively maintain the Abs’ bioactivity. In the control group, the same volume of monoclonal PSA Ab solution with different concentrations (0.2, 0.5, 1 and 2 mg/mL in the 0.01 M PBS (pH=7.4)) were directly dispensed on the T line without HFBI modification. All of the parts mentioned above were assembled sequentially on a backing card as Scheme 1a showed. Fabrication of a Test Strip Reader Device and the Procedure of Fluorescence Assay In order to accurately characterize the signals on T and C line of the FICTS, we designed a test strip reader device. The test strip reader consisted of an optic fiber spectrometer, a 405 nm laser diode, a tablet computer, and a stepper motor, as shown in Figure 4c. In this work, we carried out the fluorescence assay procedure as follows: 45 µL of PSA sample (standard sample or clinical serum) with different concentrations was added into the sample port and the sample migrated toward the absorbent pad under the capillary forces. After a certain time (15 min), the fluorescence intensity on the T and C line was recorded in our laboratory-built test strip reader. The QD-based nanoprobes were excited by the 405 nm laser diode, the electrical signal was recorded by the optic fiber spectrometer, the ratio of the fluorescence signal on T line and C line (T/C) was used to quantify the analytes (every sample was made in triplicate ). The whole running process was actuated at a stable speed by the stepper motor. The final test results were presented on the tablet computer screen.

Results and discussion 4


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Modification of PNM with HFBI

Scheme 1. Schematic diagram of the principle of FICTS platform. (a) Structure of the traditional FICTS; (b) Structure of the HFBI-modified FICTS; (c) The sample detection without the targeting molecule; (d) The sample detection with the targeting molecule.

The principle of the FICTS is based on the double Abs sandwich immunoassay as shown in Scheme 1. For the traditional FICTS, Abs were normally sprayed on the T line with random orientation and easily lost their biological activities in most cases17. Generally, the common IgG Ab composes of two heavy chains and two light chains forming a “Y” shaped structure. The top region of the “Y” is the “Fab” section, which can specifically recognize and bind to the antigens. Therefore, how to expose the “Fab” region efficiently plays an important role in the retention of Abs’ activities and the antigen detection. Herein, we focused on a kind of amphiphilic protein (HFBI) and employed them to modify the T and C line on the PNM (Scheme 1b). Our hypothesis is that the “Fc” site of the Ab is likely to be adsorbed vertically on the HFBI film. Therefore, a layer of upright “Y” shaped Abs would be formed on the HFBI-modified PNM rather than multilayer with random orientation on the HFBI-unmodified PNM.

5



Figure 1. A:Wide XPS spectra of (a1) HFBI-modified PNM and HFBI-unmodified PNM, (a2) the spectra of S2p spectra of HFBI-modified PNM, (a3) the spectra of N1s of HFBI-modified and HFBI-unmodified PNM. B: SEM images of HFBI-modified PNM with different Abs concentration. The HFBI-modified PNM contains: (b1) 0 mg/mL, (b2) 0.2 mg/mL, (b3) 0.5 mg/mL, (b4) 1 mg/mL, (b5) 2 mg/mL and (b6) 5 mg/mL Abs. SEM images of HFBI-unmodified PNM with different Abs concentration. The HFBI-unmodified PNM contains: (b7) 0 mg/mL, (b8) 0.2 mg/mL, (b9) 0.5 mg/mL, (b10) 1 mg/mL, (b11) 2 mg/mL and (b12) 5 mg/mL Abs. The scale bar is 1µm for all images. C: AFM images: (c1) a rodlet mosaic structure that was typical Ab observed on HGFI-unmodified mica, (c2) an uniform Abs layer was formed on HGFI-modified mica. The scale bar is 1000 nm for both images.

To verify the adsorption performance of HFBI on the PNM, we applied XPS which was a widely 6


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used technique for analyzing the chemical and elemental composition. Figure 1a1 showed the XPS wide spectra of the PNM surface with and without HFBI modification. Obviously, HFBI-unmodified PNM displayed its typical XPS spectra with high intensity of O1s, N1s and C1s signals, but without detectable S element. However, the obvious S2p signal was observed on the surface of HFBI-modified PNM. It is well known that HFBI contains eight conserved cysteine residues that form four intramolecular disulfide bonds30. This result indicated that HFBI protein was indeedly adsorbed and covered on the surface of PNM. Figure 1a2 showed the narrow XPS spectrum of S2p (168.5 ev) of the HFBI-modified PNM. The narrow scan N1s spectrum was presented in Figure 1a3. The peak position for nitrogen was centered at around 400.5 eV that was generated by -NH3 groups in HFBI protein, confirming again the successful adsorption of HFBI protein on the surface of PNM. After that, two kinds of Abs were subsequently sprayed onto the HFBI film to form T line and C line, at the same time, the morphological changes were also characterized between the HFBI-modified and HFBI-unmodified PNM. The characterization of SEM (Figure 1b1, 1b7) indicated that the porous property of nitrocellulose membrane was not significantly changed after HFBI modification, attributing to the thickness (about 3-10 nm) and flexility of the HFBI film31. After spraying Abs at 0.2 mg/mL (Figure 1b2), 0.5 mg/mL (Figure 1b3) and 1 mg/mL (Figure 1b4), there were no noticeable morphology changes. However, the porous surfaces became smooth after loading Abs with higher concentrations (2 mg/mL and 5 mg/mL, Figure 1b5 and b6), this is mainly due to the massive Abs filled almost all the pores or caves of the PNM. Unlike the HFBI-modified groups, the morphology changes of HFBI-unmodified PNM happened readily even with the moderate Ab concentrations (0.5 and 1 mg/mL, Figure 1b9 and b10), it may be due to that the orientation of immobilized Abs was distinct from that in the control group after HFBI modification. To further verify the effect of HFBI, AFM was employed to characterize more details about the orientation of the Abs in the test strips. Owing to the intrinsic polyporous property of PNM, it was difficult to get the details. Alternatively, we introduced the mica as the substitute for nitrocellulose membrane due to its similar hydrophilic character with PNM and its smooth surface. As shown in Figure 1c1, there were plenty of “rod-like” structures on the bare mica surface with the thickness of the immobilized IgG Abs about 50 nm. Considering the dimensions of the IgG Abs (Y-shape, height = 14 nm, width = 10 nm, thickness = 5nm)17, the result suggested that multilayer Abs formed on the hydrophilic mica surface due to protein-protein interactions. In contrast, as shown in Figure 1c2, by spraying the same concentration of IgG Abs, an uniform and compact layer of “round-like” structures was formed on the HFBI-modified mica surface with a mean thickness of 15 nm, which was consistent with the height of 7



Ab (14 nm), indicating an Ab monolayer formed on the hydrophilic HFBI-modified mica surface in the “Y” orientation16. So in the HFBI-modified PNM group, more Abs were needed to fill up all the caves due to the compact “stand-up” orientation of the Abs. In contrast, “flatted” and random orientation resulted in much less amount of Abs to smooth the pores in the control group. Optimization and Characterization of HFBI-modified FICTS As proved that IgG Abs could readily adsorb onto the HFBI-modified substrate by protein-protein interaction between Ab molecule and HFBI protein, we further optimized and tested the Abs’ activity on HFBI-modified PNM. Because the solution pH has a great impact on the protein-protein interaction20, we first studied the influence of solution pH, as shown in Figure 2a, by spraying the same concentration of IgG Abs and HFBI on PNM (T line), the fluorescent signals obviously varied with the changing of the solution pH values. When the pH value reached 10, we got a highest fluorescent signal on T line than those at other pH values under the same concentration of targets. Figure 2b recorded the T/C signal ratio about Figure 2a. It was clear that there was a highest signal ratio at pH 10, indicating the solution pH value did affect the activity of the Abs on the HFBI-modified PNM substrate. Under the optimal pH, we also found that the HFBI-modified FICTS performed higher signal intensity than those of the HFBI-unmodified FICTS under the same concentrations of Abs and PSA targets (Figure 2e-h), these indicated that the Abs immobilized on HFBI-modified PNM maintained higher bioactivity than those immobilized on HFBI-unmodified PNM. Then, we further optimized the effective reaction time of HFBI-modified FICTS. The reaction time was recorded from 5 to 30 min by using 45 µL of samples containing 0 or 12 ng/mL PSA targets. The T/C and S/N signal ratios were tested to determine the effective reaction time. As shown in Figure 2c and 2d, both the T/C and S/N reached a constant value in 15 min after samples were added, revealing that 15 min was enough for quantitative analysis in all of the succeeding studies.

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Figure 2. Optimization of HFBI-modified FICTS. Plot of T/C ratio under different pH value of Abs dilutions (the concentration of Abs on the T line is 1 mg/mL): (a) The fluorescent signal readout of HFBI-modified FICTS at different pH value with the same target concentration of PSA targets (2 ng/mL); (b) The T/C signal ratio by a test strip reader (pH=7.4, 8.0, 9.0, 10.0, 10.25). (c) The analysis of the effective immune time of HFBI-modified FICTS. The concentration of antigen was 0 ng/mL (control) or 12 ng/mL (sample); (d) The S/N ratio under different immune time. The sensitivity comparison of HFBI-modified and HFBI-unmodified FICTS with different Abs concentration: (e) 0.2 mg/mL Abs, (f) 0.5 mg/mL Abs, (g) 1 mg/mL Abs and (h) 2 mg/mL Abs.

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In order to get higher signal intensity and decrease the waste of Abs, we also optimized the Abs dosage. PSA targets with different concentration (0, 0.2, 1, 2 and 5 ng/mL) were tested by HFBI-modified and -unmodified FICTS with different concentrations of monoclonal PSA Abs (0.2, 0.5, 1 and 2 mg/mL) in T line. In Figure 2e-h, it can be observed that only with high concentration of monoclonal PSA Abs (1 and 2 mg/mL) in T line, the fluorescence signal of T/C ratio showed an obvious upward tendency as the increasing concentration of PSA targets on HFBI-unmodified FICTS. In contrast, the fluorescence signal obviously strengthened as the increasing PSA targets concentration in HFBI-modified groups, even with very low concentration of monoclonal PSA Abs (0.2 mg/mL) in T line. It may be due to the “stand-up” oriented immobilization of Abs by HFBI, which can efficiently adsorb Abs with highly exposing “Fab” region and high retention of bioactivity. For the HFBI-unmodified FICTS, more PSA Abs were needed to produce high T/C signal ratio due to the random orientation and multilayer superposition with unexposed “Fab” of Abs, which easily lost their biological activity, so only the high content PSA Abs may supply more Abs with activity. In the following experiment, we choose the low concentration (0.2 mg/mL) of monoclonal PSA Abs in T line on HFBI-modified FICTS. Sensitivity and Specificity of HFBI-Modified FICTS

10


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Figure 3. (a) Calibration curve for quantitative detection of PSA targets by HFBI-modified and -unmodified FICTS; (b) The visual readout for the detecting different concentration of PSA targets by HFBI-modified FICTS under a UV laser. (c) The selectivity of HFBI-modified FICTS toward CEA, CA153, HCG, AFP, PSA and blank control; (d) The T/C signal ratio value about (c); (e) The S/N signal ratio value about (c). The concentration of each antigen was 50 ng/mL, and the blank control with 0 ng/mL PSA sample.

Under the optimal reaction conditions, we further evaluated the sensitivity of the HFBI-modified FICTS by detecting PSA targets. Under the condition of 0.2 mg/mL of monoclonal Ab on T line, PSA targets (45 µL) with different concentrations (0, 0.2, 1, 2, 5 and 12 ng/mL) were measured in 15 min after samples were dropped on the HFBI-modified and HFBI-unmodified FICTS, respectively. Results showed that T/C signal ratio of the HFBI-modified FICTS was continuously increased with the increasing PSA targets concentration, and a linear relationship between them was observed, as described in the standard curve of Y=0.027X + 0.082 (X= PSA target concentration, R2=0.965) (Figure 3a). The LOD derived from the equation was 0.06 ng/mL, which was defined as being three times the blank control sample’s standard deviation (S/N = 3). In contrast, the LOD of control group was 2.7 ng/mL, which was derived from the 11



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standard curve of Y=0.001X + 0.035 (X= PSA targets concentration, R2=0.971). In addition, we also summarized recently published results based-on ICTS about the detection limit and the Abs concentration on T line (Table 1). By contrast, it was found that compared with various signals probes-based FICTS, HFBI-modified FICTS reached very high sensitive detection with the least dosage of Abs, which will benefit for the low cost application in both resource-rich and resource-limited settings. Table 1. The summary of the published reports based-on ICTS about the detection limit and the Ab concentration on the T line Ab

Nanoparticles

Target analytes

Detection limit

1

QDs and HFBI-modified NC

PSA

0.06 ng/mL

0.2 mg/mL

Our results

2

QDs

8 ng/mL

1 mg/mL

Li. 201032

3

QD nanobeads

PSA

3.87 ng/mL

1 mg/mL

Li. 201413

Alpha fetoprotein

3 ng/mL

1 mg/mL

2 ng/mL

1 mg/mL

201533

Aflatoxin B1

2.5 ng/mL

0.5 mg/mL

Liu. 201534

NSE

0.094 ng/mL

2.5 mg/mL

4

QDs

Nitrated ceruloplasmin

Carcinoembryonic antigen

5

FITC-doped polystyrene NPs

6

magnetic nanobeads

7

GNPs

8

GNPs

9 10

CEA

concentration

0.045 ng/mL

2 mg/mL

10 ng/mL

2 mg/mL

Human IgG

0.2 ng/mL

1 mg/mL

GNPs

Troponin I

1 ng/mL

1 mg/mL

GNPs

Abrin-a

0.1 ng/mL

1 mg/mL

alternariol monomethyl ether

Reference

Wang.

Lu. 201735 Man. 201736 Parolo. 201337 Zhu. 201138 Yang. 201139

Specificity of the immunoassay is also the key property for the application of FICTS. To determine the specificity, four groups of analytes (carchinoembryonic antigen (CEA), carbohydrate antigen 153 (CA153), human chorionic gonadotropin (HCG), and alpha-fetoprotein (AFP)) were analyzed as the negative control groups as well as PSA targets as the test group and FBS as the blank control group. Figure 3c displayed the image of the six groups in dark field under a UV laser, in which a clear distinction can be observed between the test and control groups. For the test group, an obvious fluorescent band of the T and C line was observed. The T/C (Figure 3d) and S/N (Figure 3e) signal ratios were remarkably higher than those in the blank control group. Meanwhile, almost no crosstalk or interference was observed by naked eye or detected by the test strip reader. These features provide strong evidence of the high specificity of the HFBI-modified FICTS. 12


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Clinical Test of PSA targets in Human Serum by HFBI-modified FICTS

Figure 4. The data analysis of the 150 serum samples by the HFBI-modified FICTS and the ECLIA. (a) Verify the HFBI-modified FICTS accuracy of clinical detection by plotting fluorescence intensity versus 150 serum samples with various concentrations of PSA targets by ECLIA; (b) Analysis on the accuracy of the clinical detection data; (c) Test instrument of the HFBI-modified FICTS.

As a way of point-of-care testing, clinical detection highly weighs upon its efficiency and applicability40, therefore, we designed and assembled the detection device (Figure 4c) with the portable size (30 cm* 25 cm* 20 cm), the simple operation (touching type operation) and short testing time (1 minute). Generally, when the target is present in the sample, it will interact with the QDs-based nanoprobes and be captured on the T line to generate a positive result. If the target is absent, QDs-based nanoprobes will only be bound to the C line, returning a negative result (Figure 4c). Herein, the T/C signal ratio was used to determine the amount of target in the sample. Encouraged by the outstanding HFBI-modified FICTS and advantageous detection device, we tested the accuracy and applicability of this assay by detecting 150 human serum samples (gathered from volunteers) with the PSA clinical reference value of 4 ng/mL. The study was approved by the Ethics Committee at Tianjin Medical University. Informed consents were obtained from all patients in accordance with the guidelines for conducting the clinical research. As shown in Figure 4a, the clinical samples were tested by both HFBI-modified FICTS and the commercial ECLIA, which was the golden 13



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standard method in hospital. By comparing the results from these two assays, our results revealed only 2 false negative cases, which were due to the sample concentration is too close to the clinical reference value (4 ng/mL). The validity for these serum samples by HFBI-modified FICTS was 98.6%, with R2= 0.9739. Furthermore, the test results have been statistically treated to analyze the precision of each sample. Figure 4b showed that 64 human serum samples’ absolute error value were less than 10% and 140 human serum samples’ absolute error value were less than 20% in 150 samples. The percentages were 42.67% and 93.33%, which indicated that the detection of PSA targets by the HFBI-modified FICTS in biological samples was accurate. Therefore, HFBI-modified FICTS demonstrated a promising method for accurate detection of PSA targets and especially opened up a new avenue for convenient and fast clinical applications.

Conclusion Herein, we successfully constructed a HFBI-modified PNM for effectively retaining bioactivity of low concentration Abs based on FICTS. The utilization efficiency of Abs was much higher by taking best use of the active site under limited Abs quantity, so it saves a large amount of Abs, suggesting the immobilization strategy especially meaningful for expensive biomolecules. This constructed HFBI-modified FICTS not only provided qualitative detection but also be applied in patient serum sample assay. The method is rapid (15 min), convenient and easy-operation with only 45 µL of sample. In our results, we only use 0.2 mg/mL of monoclonal Abs on T line to realize the LOD as low as 0.06 ng/mL for PSA targets detection, due to their unique advantages, it may be an ideal choice for near patient and point-of-care application.

Acknowledgments The authors gratefully acknowledge that this work was financially supported by the National key research and development plan “nano-tech” key project (2017YFA0205104), the Natural Science Foundation of Tianjin, China (14JCYBJC43500, 15JCQNJC03100, 17JCQNJC09100), the National Natural Science Foundation of China (31600800) and the State Key Laboratory of Medicinal Chemical Biology, China.

Supporting Information Available Supplementary material (including the TEM and fluorescent spectra of water-soluble quantum dots) is available.

Notes 14


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The authors declare no competing financial interest.

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Table of Contents Graphic:

In this work, a novel amphiphilic hydrophobin (HFBI) protein was introduced to modify the test (T) line of FICTS to effectively immobilize and retain the antibodies’ bioactivity in the “stand-up” orientation. (a) The scheme of the traditional FICTS, in which the antibodies were normally sprayed on the T line with random orientation. (b) The scheme of the HFBI-modified FICTS, in which the HFBI were employed to modify the T and C line and a layer of upright “Y” shaped antibodies would be formed on the HFBI-modified FICTS.

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Effective Bioactivity Retention of Low Concentration Antibodies on

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