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Reverse Fluorescence Enhancement and Colorimetric Bimodal Signal Readout Immunochromatography Test Strip for Ultrasensitive Large-Scale Screening and Postoperative Monitoring Yingyi Yao, Weisheng Guo, Jian Zhang, Yudong Wu, Weihua Fu, Tingting Liu, Xiaoli Wu, Hanjie Wang, Xiaoqun Gong, Xing-Jie Liang, and Jin Chang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b08445 • Publication Date (Web): 22 Aug 2016 Downloaded from http://pubs.acs.org on August 23, 2016

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ACS Applied Materials & Interfaces

Reverse

Fluorescence

Colorimetric

Bimodal

Immunochromatography Ultrasensitive

Enhancement Signal Test

Large-Scale

and Readout

Strip

for

Screening

and

Postoperative Monitoring Yingyi Yao,a,§ Weisheng Guo,b,§ Jian Zhang,a Yudong Wu,a Weihua Fu,c Tingting Liu,c Xiaoli Wu,a Hanjie Wang, a Xiaoqun Gong,a* Xing-jie Liang,b and Jin Chang a* a School of Materials Science and Engineering, 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 Laboratory of Controllable Nanopharmaceuticals, CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology, Beijing 100190, China c Department of General Surgery, Tianjin Medical University General Hospital, Tianjin 300052, China § Y.Y. and W.G. have equal contribution to this paper.

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

ACS Paragon Plus Environment


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Abstract Ultrasensitive and quantitative fast screening of cancer biomarkers by immunochromatography test strip (ICTS) is still challenging in clinic. The gold nanoparticles (NPs) based ICTS with colorimetric readout enables a quick spectrum screening but suffers from nonquantitative performance; although ICTS with fluorescence readout (FICTS) allows quantitative detection, its sensitivity still deserves more efforts and attentions. In this work, by taking advantages of colorimetric ICTS and FICTS, we described a reverse fluorescence enhancement ICTS (rFICTS) with bimodal signal readout for ultrasensitive and quantitative fast screening of carcinoembryonic antigen (CEA). In the presence of target, gold NPs aggregation in T line induced colorimetric readout, allowing on-the-spot spectrum screening in 10 min by naked eye. Meanwhile, the reverse fluorescence enhancement signal enabled more accurately quantitative detection with better sensitivity (5.89 pg/mL for CEA), which is more than 2 orders of magnitude lower than that of the conventional FICTS. The accuracy and stability of the rFICTS were investigated with more than 100 clinical serum samples for large-scale screening. Furthermore, this rFICTS also realized postoperative monitoring by detecting CEA in a patient with colon cancer and comparing with CT imaging diagnosis. These results indicated this rFICTS is particularly suitable for point-of-care (POC) diagnostics in both resource-rich and resource-limited settings.

Keywords: POCT, Reverse Fluorescence Enhancement ICTS (rFICTS), Carcinoembryonic Antigen (CEA), Bimodal Signal Readout, Postoperative Monitoring


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Introduction Point-of-care testing (POCT) is becoming a clinical-medicine discipline that evolves rapidly in analytical scope and clinical application.1-3 Different from the laboratory medicine, the POCT are particularly suitable for on-the-spot patient care as user-friendly approaches, owing to the minimized portable instrument, easy-to-use, short turnaround time as well as cost-effective, which greatly benefits the prompt treatment and development of medical diagnosis in resource-poor setting. As the most common commercial POCT format, immunochromatography test strip (ICTS) based on gold nanoprobes has been widely applied into the field of the pathogens, malignant tumors and cardiovascular diseases detection, benefitting from the short time consuming, small sample volumes, visible signal readout and no need of incubation, washing steps and professional training operation.4-5 Despite of the distinguishing analytical performance, ICTS only allows qualitative or semiquantitative analyzing detection with poorer sensitivity compared with other molecular techniques such as enzyme-linked immunoassay (ELISA) and electrochemiluminescence immunoassay (ECLIA). The signal generating reagents have great impact on the detection sensitivity and quantitative of ICTS. Various fluorophors including up-converting phosphor,6 organic dyes,7 gold clusters8 and quantum dots9-11 have been widely exploited as fluorescence signal generator to fabricate fluorescence ICTS (FICTS), which showed advantageous features of high sensitivity and easy quantitative detection over the conventional colorimetric ICTS.12 In our previous work,13-14 quantum dots based FICTS device has been successfully fabricated for quantitative detection of alpha fetoprotein (AFP) and prostate specific antigen (PSA) with sensitivity of 1 ng/mL and 0.33 ng/mL, which is about 10 times lowers than that of gold NPs based colorimetric ICTS. Although great efforts have been made on improving the analysis sensitivity of ICTS, it is still far lower than that of the common clinical detection approaches, (such as ELISA and ECLIA), thus easily results intense concerns about the reliability of ICTS applications in the



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early diagnostics and postoperative monitoring of disease. Additionally, the FICTS abandoned the straightforward visible readout and thereby was deprived of the large-scale fast screening merit of colorimetric ICTS. Thus, it’s still an unmet challenge for ICTS to realize ultrasensitive and quantitative quick screening of cancer biomarkers with ultralow concentrations in clinical samples. Organic dyes adapted for FICTS, for example Cy5 exhibits strong fluorescent brightness benefitting sensitive and quantitative detection, but suffer from easy quenched performance. On the other hand, owing to the high surface area-to-volume ratio, greatly efficient fluorescence quenching capability and aggregation induced color emergence, gold nanoparticles have been intensely assessed for ICTS, which allows quick naked eye readout for spectrum screening. Herein, by taking advantage of the distinctive properties of dyes and gold NPs, we designed a novel reverse fluorescence enhancement ICTS (rFICTS) with bimodal signal readout for detection of carcinoembryonic antigen (CEA). This proposed rFICTS approach presented advantageous features of quick large-scale screening by colorimetric readout and sensitive detection of cancer biomarker with ultralow concentrations by reverse fluorescence enhancement. As showed in Scheme 1, this novel ICTS approach bears a control line made of bare IgG, which can capture the detection antibody (Ab1) without antigen, and a test line with capture antibody (Ab2) decorated Cy5 printed as fluorescent probes. After applying sample to the lateral flow strip, Ab1 decorated AuNPs immigrated to the control line alone with a sample. In the presence of target antigen (CEA), AuNPs aggregated in the test line based on specifically sandwich-like immunological reaction, which resulted colorimetric readout and significant fluorescence quenching against Cy5. Different from the previously reported FICTS assays focused on increasing dye-to-Ab1 ratio, this proposed rFICTS approach intensely depends on the great fluorescence quenching efficiency of AuNPs, enabling more accurately quantitative detection with better sensitivity (5.89 pg/mL for CEA) than conventional FICTS assays, which is


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comparable to the gold standard method ELISA in clinic. Meanwhile, this rFICTS assay also provides an alternative readout modality to the reverse fluorescence enhancement. AuNPs aggregation induced color emergence allowed on-the-spot naked eye readout when the concentration of CEA is higher than the normal level, which benefits a quick spectrum screening. Additionally, CEA as a broad-spectrum cancer marker could reflect the existence of a wide variety of cancer in clinic. This proposed approach cannot only achieve ultrasensitive and quantitative large-scale screening with more than 100 patient serum samples, but also realize postoperative monitoring by detecting CEA in a patient with colon cancer and comparing with CT imaging diagnosis. These results indicated the great potential of this rFICTS in real-world applications.

Results and discussion Mechanism of the rFICTS. In this work, rFICTS with bimodal signal readout were successfully constructed based on the nanoparticle surface energy transfer (NSET) effect and aggregation induced coloration of gold nanoparticles, as illustrated in Scheme 1. Cy5 dyes were multiply labelled on the capture antibody (Ab2) as the donors, and detection antibody (Ab1) decorated AuNPs were applied on the ICTS as the energy acceptors and nanoprobes. In the presence of CEA, the two components were in close proximity by the sandwich-like immunoassay, which resulted aggregation of AuNPs on T line and concomitant significant fluorescence quenching against Cy5 via the NSET effect. Thereby this versatile trip achieved reverse fluorescence enhancement and colorimetric bimodal signal readout, realizing ultrasensitive and quantitative detection and quick large-scale screening, respectively. In order to achieve reverse fluorescence enhancement, the donor-acceptor pair was selected based on careful considerations. AuNPs have been widely demonstrated as ultra-efficient quenchers of molecular excitation energy (up to 99.8%) with a long quenching range (more than 40 nm distance)15 through the NSET effect. Cy5 was chosen as



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the donor to construct excellent chromophore-AuNP composites due to its near infrared fluorescence to attenuate the autofluorescence of biomarkers.16-18 Our results in Figure 1 indicated the high fluorescence of Cy5 was efficiently quenched in the presence of AuNPs.19-21 The AuNPs size played important roles on fluorescence quenching profile and lateral immigration, and thereby influenced the test strip performance. It has been well proved that particles with size less than 50 nm showed high scattering intensity owed to the Mie extinction coefficient.22 Therefore, the fluorescence quenching profile of AuNPs with various sizes (20 nm, 30 nm and 40 nm) was investigated by incubation with Cy5 solution. The results in Figure 1b indicated the positive correlation between the quenching efficiency and AuNP size.23-25 Additionally, Figure S1 (see Supporting Information) revealed that the Cy5 concentration was exactly linearly proportional to the fluorescence intensity, suggesting quantitative detection by fluorescence intensity. The lateral immigration and aggregation induced coloration behavior of the AuNPs were studied on the strip.26-27 It has been widely concluded that AuNPs with larger size were suffered from poor colloidal stability induced nonspecific absorbance and incomplete release on the test pad.28-29 As showed in Figure 1d, the AuNPs with size of 40 nm resulted serious retention of nanoparobes at the end of the conjugate pad on the strip, while the 30 nm AuNPs could avoid this shortage and induce obvious colorimetric readout as well. Based on the comprehensive considerations of fluorescence quenching ability and lateral immigration behavior of AuNPs, the 30 nm AuNPs were selected as the optimal candidate for rFICTS establishment. The established rFICTS were distinct with the ultrasensitive detection limit, which should profit from the superior fluorescence quenching ability of AuNPs. For deep insight of the detection mechanism, the Cy5 dyes were directly sprayed onto the square nitrocellulose membrane with the length of 5 mm, followed by the AuNPs droplets. To guarantee the accuracy of the experiment, the dosage of the Cy5 and the AuNPs were fixed same with that of actual


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reaction of the rFICTS. By comparing the change of fluorescence intensity before and after reaction (Figure 1c), the quenching efficiency of the gold nanoparticle was estimated to be 2320 Cy5 molecules each AuNP. (see Eqn.1-3 in the Supporting Information) Sensitivity, Selectivity and Stability of the rFICTS. Under the optimal conditions, we further evaluated the sensitivity, selectivity and stability of the rFICTS by detecting CEA. CEA standards aliquots (50 µL) with concentration ranging from 1 to 106 pg/mL were tested 10 min by the rFICTS and the conventional fluorescence ICTS, respectively. Results showed that the changing value of fluorescence intensity (∆F) on T line continuously increased with the CEA concentration increasing from 10 to 106 pg/mL (Figure 2). A linear relationship between ∆F and c(CEA) was observed in the range between 10 and 103 pg/mL, as descripted in equation of Y=74238.79X + 257811.42 (X= log[CEA concentration], R2=0.963) (Figure S4). The LOD derived from the equation was 5.89 pg/mL, which was 2 orders of magnitude lower than that of the FICTS with the LOD about 1000 pg/mL. The much better detecting sensitivity of the rFICTS over the conventional FICTS should be attributed to the reverse fluorescence enhancement. In the case of FICTS, the antigen was captured via the sandwich immunoreaction, and concentration of the detected antigen was intensely related to the fluorescence signal originating from the fluorescein labeled detection antibody. Limited by the 3-D orientation of antibody, a small number of fluoresceins were labeled onto each detection antibody, and the signal amplification was restricted. On the other hand, in the case of rFICTS, where capture antibody decorated with fluoresceins were printed onto the T line and AuNPs conjugated with detection antibody were applied as nanoprobes, AuNPs were anchored and aggregated on T line in the presence target via sandwich-like immunoreaction, inducing efficient quenching against all the fluoresceins within their quenching range. As described above, the quenching efficiency could reach more than 2000 fluoresceins each AuNP, resulting sighificant signal change even at a very low concentration of target analyte



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and achieving ultrasensitive and quantitative detection. The detection selectivity of the rFICTS was evaluated on other 4 tumor biomarkers, including prostate specific antigen (PSA), alpha fetoprotein (AFP), breast cancer antigen (CA15.3) and breast cancer antigen (CA12.5). As showed in Figure 3, only in the presence of CEA were the remarkable reverse fluorescence enhancement and colorimetric readout found, suggesting the high detection specificity of the rFICTS to CEA. The assay stability was determined with CEA standards at 10 and 100 ng/mL in one month. The rFICTS was sealed with mini dropper and desiccant (Figure 4C), and made each 3 repeat tests for CEA standards at 10 and 100 ng/mL at 0, 3, 7, 15, 30 days. In Figure 4A and B, the results indicated that, as time went on, the detection based on rFICTS still maintained a high accuracy and stability. The concentrations of total 15 repeat tests for CEA standards at 10 and 100 ng/mL varied by 7.23% and 7.13% of coefficient of variance (CV), respectively (Figure. 4D). Operability and Large-Scale Screening of the rFICTS. Operability and applicability as crucial factors have to be carefully evaluated from the perspective of pre-clinical and clinical research, especially for the application in source-poor setting. For the rFICTS system, AuNPs served not only as an efficient fluorescence quencher benefitting the amplified the reverse fluorescence enhancement, but also as a colorimetric signal based on aggregation induced coloration for the visual qualitative detection. Thus, as illustrated in Figure 5, with the aid of rFICTs, the targets analysis can be achieved in two manners for ultrasensitive qualitative detection and quick large-scale screaming by naked eyes, respectively. The fluorescence quantification with ultrahigh sensitivity by reverse fluorescence enhancement signal (RFES) is conducive to the early diagnosis and monitoring of disease. Meanwhile, the rapid qualitative detection with naked eye readout is of great importance for large scale screening, which is essential for timely clinical decision making and management of epidemics of


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infectious diseases in resource-constrained settings. In brief, with the advantages of bimodal readout, this proposed assay principle of the rFICTs holds great potentials as an universal POCT approach for disease screening in community and cancer diagnosis in hospital laboratory. Encouraged by the outstanding sensitivity and advantageous bimodal readout, we tested the accuracy and applicability of this assay by detecting CEA in 120 human serum samples (gathered from Tianjin Medical University General Hospital) with the CEA cutoff threshold of 5 ng/mL (clinical reference value of CEA). As showed in Figure 6, the clinical samples were tested by rFICTs and the commercial ECLIA in hospital as the golden standard method, repsectively. By comparing the results from the two assays, our results revealed only 2 false positive cases and 1 false negative case, which is due to the ultrasensitive detection sensitivity of rFICTs. It’s noticed that the accuracy of rFICTs reached 100 % when detected based on the colorimetric readout modality by naked eye. As summarized in Table 1, the validity for these serum samples by rFICTS were all over 95% (the specificity of 97.4% and 100% and the sensitivity 95.7% and 100% for CEA by RFES calculation and naked-eye readout), which indicated that the detection of CEA at the rFICTS in biological samples was accurate. Therefore, rFICTS demonstrated promise for accurate detection of CEA cancer biomarker and especially open up a new avenue for convenient and fast clinical applications. Postoperative Monitoring of the rFICTS. Tumor recurrence is the major threat for the postoperative cancer patients.30 At present, the majority of follow up methods to monitor the recurrence including the tumor biomarker test, enhanced CT or MRI scan. Usually, the patients need to go to the hospital to receive the testing or scan. Due to the radiation risk and high cost, it is impractical for patients to receive CT or MRI scan too frequently, especially for the weak patients in poverty-stricken areas. As a result the low cost POCT that could proceed at home or in the community clinic will be a promising method to monitor the tumor biomarker.



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In this work, we further applied the ICTS in postoperative detection so as to accurately monitor the condition of colon cancer patients before and after the surgery by rFICTS contrasting with the effectiveness of ECLIA and CT scan (Figure 7). Since the permission from the patients and their families is requested for this experiment, only one clinic case was monitored throughout one year postoperation in our work. Results showed that the CEA level decreased dramatically after the operation, but still remained above the upper limit of normal level, since there were some lymph nodes metastasis around aorta abdorminalis as found in the enhanced CT scan one month post operation. Following the proceeding of chemotherapy, the CEA concentration dropped to normal level and the follow-up enhanced CT scan showed that the lymph nodes metastasis around aorta abdorminalis disappeared two months after operation. The CEA value tested by rFICTS was consistent with that by ECLIA. These results proved the successful application of postoperative monitoring for colon cancer patient, and it will be a promising analysis method with low cost and convenient operation as a POCT.

Conclusion In summary, we successfully developed a reverse fluorescence enhancement ICTS approach with bimodal signal readout for ultrasensitive and quantitative fast screening of carcinoembryonic antigen (CEA). This proposed rFICTS approach has advantageous features of quick spectrum screening by colorimetric readout and sensitive detection of cancer biomarker with ultralow concentrations by reverse fluorescence enhancement. According to our results, in the presence of target, AuNPs aggregation in T line induced colorimetric readout, allowing on-the-spot spectrum screening in 10 min by naked eye. Meanwhile, the reverse fluorescence enhancement signal enabled more accurately quantitative detection with better sensitivity (5.89 pg/mL for CEA), which was more than 2 orders of magnitude lower than that of the conventional FICTS. The accuracy and stability of the rFICTS were investigated with 120 clinical serum samples for


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large-scale screening. There were only 2 false-positive and 1 false-negative cases by our assay. Furthermore, this rFICTS also successfully realized postoperative disease development monitoring by detecting CEA in a patient with colon cancer. The detection results by rFICTS were well consistent with the ECLIA and CT imaging diagnosis. Our results suggested this rFICTS has great potentials in real-world application.

Experimental Section Materials. Sucrose was purchased from Aladdin Industrial Corporation. Chloroauric acid, Sodium citrate tribasic dihydrate and potassium carbonate were purchased from Tianjin Jiangtian Chemical Co., Ltd. Polyethylene glycol (PEG) 20,000, Polyethylene glycol (PEG) 4,000 and Polysorbate 20 were purchased from Alfa Aesar (China) Chemical Co., Ltd. Bovine serum albumin (BSA) were purchased from Beijing Dingguo Biotechnology Co., Ltd. Fetal bovine serum (FBS) were purchased from GE Healthcare Life Sciences HyClone Laboratories. And the above substances were used without further purification. Sulfo-Cyanine5 NHS ester was purchased from LiTTLE-PA Sciences Co., Ltd. Carcino Embryonie Antigen (CEA) standard substance, the corresponding anti-CEA monoclonal antibodies (including detection antibodies and capture antibodies) and anti-detection antibodies were acquired from 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. Signal acquisition device - Azure C600 was purchased from the Azure Biosystems, Inc. Automatic dispenser-HM3030 was purchased Shanghai Kinbio Tech Co.,Ltd. The Preparation of Gold Nanoparticles. To prepare AuNPs (20, 30 and 40 nm), 150 mL of 2.2 mM trisodium citrate solution was mixed with 1 mL of 24 mM hydrogen tetrachloroaurate (III) hydrate (HAuCl4·3H2O) solution at 100



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ºC for 10 min under vigorous stirring. The resulting wine-red solution was then maintained at 90 ºC after being mixed with 53 mL of deionized water. Then, 55 mL of above solution was mixed with 2 mL of 60 mM trisodium citrate solution and was added to 1 mL of 24 mM HAuCl4 solution at 90 ºC for 30 min to increase AuNP size (20 nm). The above steps were repeated to fabricate AuNPs with larger sizes (30 and 40nm). All of the as-prepared particles were then washed by centrifugation in order to extract the surfactant and redispersed in deionized water. Preparation of Au-anti CEA IgG Probes. Labeling of AuNPs with CEA detection antibodies was carried out as following: To adjust the pH valves of the AuNPs solution to 8.2-8.4, 75 µL of 10 mg mL-1 K2CO3 was added in 1 mL AuNPs solution. And 7.5 µg (1 µL of 7.5 mg mL-1) CEA detection antibodies were added in it for 30 min, and then 100 µL of 10 mg mL-1 BSA and 100 µL of 1% PEG20000 were added in the AuNPs mixture for additional 15 min. Lastly the AuNPs mixture was centrifuged at 13,000 rpm for 30 min at 4 °C, and the supernatant was discarded. The resulting pellet was re-suspended in 30 µL of remnant solution for use in downstream experiments. Preparation of Cy5-anti CEA IgG Probes. First, 100 µg CEA capture antibodies was dissolved in 500 µL of 0.1M pH 8.5 borate saline buffer. One milligram of Sulfo-Cyanine5 NHS ester was dissolved in 1 mL of anhydrous dimethyl sulfoxide (DMSO), 3 µL of it was added in the CEA caputure antibodies mixture. Then the mixture was rotated in dark place for 2h. At last, the mixture was filtrated at 14000 g for 20 min at 4 °C three times. The resulting pellet was re-suspended in 40 µL of remnant solution for use in downstream experiments. Preparation of Immunochromatography Test Strip Based on the Au-anti CEA IgG Probes and Cy5- anti CEA IgG Probes. The test strip consists of a sample pad, conjugate pad, reaction membrane, absorbent pad, and backing card. The sample pad was pretreated with blocking buffer (pH 8.0, containing 20 mM


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tris (hydroxymethyl) aminomethane-HCl (TrisHCl) and 0.5% (w/v) Tween-20) and dried at 37 °C for 2 h. The conjugated pad was pretreated with blocking buffer (pH 7.4, containing 0.01 M PBS, 5% (w/v) BSA, 5% (w/v) sucrose, 1% (w/v) PEG4000, and 0.1% (w/v) Tween-20) and dried at 37 °C for 2 h. After that, the Au−anti CEA IgG probe solution were dispensed onto the conjugated pad, by ratio of 15 µL: 1 cm, and dried at 37 °C for 1h. Using the dispenser (XYZ-3050 BioJet Quanti 3000), the desired volume of Cy5-anti CEA IgG probes solution (1 mg/mL) and goat anti-mouse IgG (1 mg/mL) was dispensed on the reaction membrane to form the test zone and control zone, by ratio of 40 µL: 100 test strips, respectively. After 2 h of drying at 37 °C, all of the parts mentioned above were assembled on a plastic adhesive backing card, which was cut into 3 mm strips and stored at room temperature. Immunodetection of CEA by rFICTS and FICTS Based on Cy5-anti CEA IgG Probes. 50 µL of serum sample (or CEA standards) was added into the sample port on the rFICTS and FICTS and immune chromatography at 37 °C under the conditions of protection from light for 10 minutes. Then the strips were transferred to the signal acquisition device - Azure C600, and were exposed for 50 milliseconds. The signal of fluorescence intensity on the Test Line was read. The variation of the fluorescence intensity on the Test Line was calculated through measuring the integral density by Adobe Photoshop. Treatment of the Clinical Serum Samples. In order to test the accuracy of rFICTS for clinical application, 120 patients’ serum samples were collected from Tianjin Medical University General Hospital for CEA detecting. The procedure was approved by the Hospital Ethics Committee and each patient was signed informed consent before blood drawing. After the blood sample was drawn from the patient using a separating gel tube that containing coagulant, the serum was collected by centrifugation and stored at -80 °C for further test. Process of Post-operative Monitoring.



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Tumor recurrence is the major threat for the postoperative cancer patients. In this study, we also applied the rFICTS in postoperative detection to accurately monitor the condition of colon cancer patients before and after the surgery and chemotherapy by rFICTS contrasting with the effectiveness of ECLIA and enhanced CT scan. The study was also approved by the Hospital Ethics Committee and the patient informed consent was signed. Before the operation, the patient received enhanced CT scan of abdomen, and the detection of CEA in the blood sample was performed by rFICTS and ECLIA. One month after the operation, and before the chemotherapy, the enhanced CT scan of abdomen and the blood sample detection were also employed to evaluate the patient condition. Then, after three times of chemotherapy, the enhanced CT scan of abdomen and three times of blood sample detection were again applied to evaluate therapeutic effect.

Acknowledgments The authors gratefully acknowledge that this work was financially supported by the National Natural Science Foundation of China (51373117, 51303126, 31401578), Tianjin Natural Science Foundation(15JCQNJC03100).

Supporting Information Available Supplementary material (including TEM, UV-visible absorption spectra and DLS results for AuNPs with different particle size, the fluorescent spectra of the Cy5 solution addition of the AuNP with different sizes, the UV-vis absorption spectra of the AuNP and the AuNP conjugated antibody, the fluorescence spectra of the Cy5 and the Cy5 conjugated antibody, the linear range of CEA by the rFICTS, the hydrodynamic diameter of Au probes in serum system within 16 hours, the formula of the number of antibodies on one Au probe and the formula of the quenching efficiency of the gold nanoparticle) is available.

Notes


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Scheme 1. Mechanism of the conventional FICTS and the rFICTS for antigen detection.

Figure 1. (a) Bright-field and dark-field images of solutions containing Cy5, AuNP and Cy5 with AuNP. (b) Mix the Cy5 and same concentration of AuNPs with the diameter of 20, 30, 40 nm and calculate the quenched percentage of fluorescence intensity and the quenched Cy5 molecular number by single AuNP, respectively. (c) Fluorescence images of solutions containing Cy5 and Cy5 with AuNP on the nitrocellulose membrane. (d) Bright-field images of ICTS by the same concentration of AuNPs with diameter of 20, 30, 40 nm, respectively.



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Figure 2. Conventional FICT and the rFICT for sensing various concentrations of CEA. (a) Upside: Fluorescence images of the commonly used FICTS for various concentrations (0.01 ng/mL to 1000 ng/mL) of CEA using Ab2-Cy5 as detection tags. Down side: Fluorescence images of the newly rFICTS for the same concentrations of CEA. (b) Comparison of the detection performance of the two immunochromatography test strip by plotting fluorescence intensity versus various concentrations of CEA standards. (c) The linear range of (b). ∆F = | F0-F | was used to measure the variations of the fluorescence intensity, where F0 is the fluorescence intensity before reaction and F0 is the fluorescence intensity after reaction. Error bars show standard deviations. (n = 3)


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Figure 3. (a) The analysis of the test time of the rFICTS. Qualitative detection by naked eye after two minutes and qualitative detection by fluorescence signal after 10 minutes when fluorescence intensity is stable. The concentration of antigen was 50 ng/mL. (b) Selectivity of the rFICTS. The concentration of each antigen was 50 ng/mL.

Figure 4. The characterization of the rFICTS’ time stability. (a) 3 repeat tests for CEA standards with 10 ng/mL at 0, 3, 7, 15, 30 days by rFICTS. (b) 3 repeat tests for CEA standards with 100 ng/mL at 0, 3, 7, 15, 30 days by rFICTS. (c) The sealed package of rFICTS. (d) data plots of assay variability tests using CEA standard at 10 ng/mL and 100 ng/mL.



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Figure 5. The method of application of the bimodal test rFICTS for clinical detection.


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Figure 6. The data analysis of the 120 serum samples by the bimodal test rFICTS in large-scale screening. (a) Verify the rFICTS accuracy of clinical detection by naked-eye readout. Left side: the rFICTS with CEA standard substance as the standard values. Right side: the rFICTS with 20 serum samples from patients, the concentration of CEA by ECLIA was shown on each samples. (b) Verify the rFICTS accuracy of clinical detection by plotting fluorescence intensity versus 100 serum samples with various concentrations of CEA by ECLIA. ∆F = | F0-F | was used to measure the variations of the fluorescence



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intensity, where F0 is the fluorescence intensity before reaction and F is the fluorescence intensity after reaction.

Table 1. Clinical data analysis of CEA by using the rFICTS

By RFES Calculation By Naked-eye Readout

Sample Size

Tested Positive

Tested Negative

Validity

77 Positive

75

2

Specificity 97.4%

23 Negative

1

22

Sensitivity 95.7%

15 Positive

15

0

Specificity 100%

5 Negative

0

5

Sensitivity 100%

Figure 7. (a) The concentration of CEA tested by rFICA and ECLIA at pre-operation and post-operative follow-up. (b) The CT images of the colon neoplasm at pre-operation and post-operative follow-up. (c) The CT images of the lymph node at pre-operation and post-operative follow-up.


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