Homogeneous Immunosorbent Assay Based on Single-Particle

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Homogeneous Immunosorbent Assay based on SingleParticle Enumeration Using Upconversion Nanoparticles for the Sensitive Detection of Cancer Biomarkers Xue Li, Lanlan Pan, Lin Wei, Zunyan Yi, Xiao Wang, Zhongju Ye, Lehui Xiao, Hung-Wing Li, and Jianfang Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00251 • Publication Date (Web): 20 Mar 2018 Downloaded from http://pubs.acs.org on March 21, 2018

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Analytical Chemistry

Homogeneous Immunosorbent Assay based on Single-Particle Enumeration Using Upconversion Nanoparticles for the Sensitive Detection of Cancer Biomarkers Xue Li,1,2 Lin Wei,2 Lanlan Pan,2 Zunyan Yi,2 Xiao Wang,1 Zhongju Ye,1 Lehui Xiao,*,1 Hung-Wing Li,*,3 and Jianfang Wang4 1

State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of

Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin, 300071, China; 2

Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research,

Ministry of Education, Key Laboratory of Phytochemical R&D of Hunan Province, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha, 410081, China; 3

Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong,

SAR China; 4

Department of Physics, The Chinese University of Hong Kong, Shatin, Hong Kong,

SAR China. *

Corresponding authors

Email: [email protected], [email protected] Fax: +86-022-23500201

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Analytical Chemistry 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

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ABSTRACT

Prostate-specific antigen (PSA) is an intercellular glycoprotein produced primarily by the prostate gland, which is commonly chosen as the initial target for the early diagnosis of prostate cancer. In this work, we demonstrate a simple yet sensitive sandwich-type single-particle enumeration (SPE) immunoassay for the quantitative detection of PSA in a flow chamber. The design is based on the luminescence resonance energy transfer (LRET) between upconversion nanoparticles (UCNPs) and gold nanoparticles (GNPs). The carboxyl group-functionalized UCNPs are conjugated with anti-PSA detection antibodies (Ab1) and serve as the luminescence energy donor, while GNPs are modified with anti-PSA capture antibodies (Ab2) and act as the energy acceptor. In the presence of target antigen (i.e., PSA), the specific immnuoreaction brings the donor and acceptor into close proximity, resulting in quenched luminescence. Through statistical counting of the target dependent fluorescent particles on the glass slide surface, the quantity of antigens in the solution is accurately determined. The dynamic range for PSA detection in Tris-buffered saline (TBS) is 0-500 pM and the limit-of-detection (LOD) is 1.0 pM, which is much lower than the cut-off level in patients’ serum samples. In the serum sample assay, comparable LOD was also achieved (i.e., 2.3 pM).

As a

consequence, this method will find promising applications for the selective detection of cancer biomarkers in clinical diagnosis.

KEYWORDS

Upconversion nanoparticles; Gold nanoparticles; Prostate-specific antigen; Cancer biomarker; Single particle enumeration 2 ACS Paragon Plus Environment

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INTRODUCTION

Prostate cancer, a major cancerous disease in elderly males, accounts for about 10% of deaths from cancers.1,2 The accurate and early diagnosis of the disease in the early stage may save millions of lives. Prostate-specific antigen (PSA) is a neutral serine protease and is an intercellular glycoprotein (34 kDa kallikrein-like protease) produced primarily by the prostate gland.3 The level of serum PSA in the range of 60-90 pM is considered of high risk in suffering from prostate carcinoma within 15 years.4 Hence, PSA, as an oncological biomarker, is chosen as the initial target for prostate cancer diagnosis. PSA levels are usually determined via immunoreaction in clinical applications. Up to now, although many detection methods, including the fluorescence spectroscopic measurement,5 enzyme-linked immunosorbent assay (ELISA)6 and electrochemical assay7 have been established, most of them suffer from the limitations such as low sensitivity and long operation time. A rapid, sensitive and convenient strategy for the detection of PSA is thus highly desirable. Recently, single-particle (or molecule) enumeration (SPE) has attracted more and more attention for clinical biomarker detection because they are well-equipped to provide excellent sensitivity and high resolution that is not achievable by ensemble measurements.8-16 Until now, many fluorescent biolabels have been reported for SPEbased cancer biomarker detection including biological (e.g. phage nanofibers and nanoparticles)17-20 and non-biological nanoparticles (e.g. quantum dots, organic dyes, and other fluorescent nanoparticles)21-26. However, the intrinsic optical property of these 3 ACS Paragon Plus Environment


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materials and significant autofluorescence from biological samples usually result in limited detection sensitivity and low signal-to-noise ratios. Nonetheless, photonupconversion fluorescent nanoparticles-based (UCNPs, lanthanide-doped nanomaterials) optical sensing has drawn great attention because of its superior optical properties, including high quantum yields, large anti-Stokes shifts, sharp emission bandwidth, low toxicity, good photostability and high chemical stability.27,28 Particularly, the deep NIR light excitation enables the effective screening of the background fluorescence from biological samples, which is therefore suitable for single-particle imaging in a complex biological environment.29-33 Many sensors have been developed based on UCNPs, for example, luminescence resonance energy transfer (LRET)-based immune sensing where UCNPs are served as the donor for spectroscopic measurements.5,34-38 In this work, we developed a homogeneous LRET-based SPE method for the quantitative detection of PSA in solution, as illustrated in Scheme 1. The UCNPs conjugated with the detection antibodies (UCNPs-Ab1) served as the fluorescence donor. In the absence of the target molecules, the capture antibodies-functionalized quencher (gold nanoparticles, GNPs-Ab2) would not specifically bind toward the UCNPs-Ab1. Taking advantage that LRET is a distance-dependent process, only when the donor and acceptor are located close enough, the fluorescence from the donor can then be effectively quenched. In the presence of the target antigens, the immunoreaction-induced particle-particle association could specifically drag the donor and acceptor together, resulting in a dosage-dependent fluorescence quenching process. Through counting the concentration-dependent fluorescent particles on the glass slide surface, a dynamic range of 0-500 pM for PSA detection was realized with a LOD of 1.0 pM in Tris-buffered 4 ACS Paragon Plus Environment

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saline. Comparable LOD was also achieved in serum sample (i.e., 2.3 pM). Owing to the high specificity of the immunoreaction, no interference was observed from other protein molecules such as IgG and HSA. Therefore, the developed strategy herein will find broad applications for SPE-based immune analysis in complex surroundings in the future.

EXPERIMENTAL SECTION Materials and Instruments. Ytterbium(III)

chloride

hexahydrate

(YbCl3·6H2O),

Yttrium(III)

chloride

hexahydrate (YCl3·6H2O), erbium(III) chloride hexahydrate (ErCl3·6H2O), oleic acid (OA),

1-octadecene

(1-ODE),

1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide

hydrochloride (EDC), N-hydroxysuccinimide (NHS), polyacrylic acid (PAA, MW: 2000), chloroauric acid (HAuCl4), methanol (CH3OH), 3-aminopropyltriethoxysilane (APTES), bovine serum albumin (BSA), and human serum albumin (HSA) were purchased from Aladdin (Shanghai, China). Thiolated methoxyl-polyethylene glycol (CH3O-PEG-SH, MW: 6000) were purchased from Sigma-Aldrich (U.S.A.). Sodium citrate, sodium hydroxide (NaOH), ammonium fluoride (NH4F), cyclohexane, ethanol (CH3CH2OH), phosphate buffered saline (PBS) solution, sodium chloride (NaCl), potassium chloride (KCl), and Tris-HCl were obtained from Sinopharm Group (Shanghai, China). Human IgG, anti-PSA capture antibody, and anti-PSA detection antibody were purchased from Shanghai Linc-Bio Science Co. Ltd. (Shanghai, China). All other reagents were of analytical purity. Real serum samples were obtained from a healthy volunteer. A modified upright optical microscope (Olympus, Japan) was utilized for the singleparticle imaging experiments. The laser beam from a fiber coupled with 980 nm cw laser 5 ACS Paragon Plus Environment


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was expanded with a collimating lens and projected to the back port of the filter cube. The light was then reflect by a short-pass dichroic mirror and focused at the back focal plane of the objective. The emitted fluorescence from the UCNPs was collected by the objective and filtered with a band-pass filter. A Hamamatsu CMOS camera was used to capture the image for the SPE measurements. The upconversion fluorescence spectra from UCNPs were measured by a fluorescence spectrometer equipped with a 980 nm cw laser. The absorption spectra of GNPs were recorded using spectrophotometer (UV-2450, Japan). The size distributions and zeta potential of UCNPs and GNPs were measured with a Zetasizer Nano ZS system (Malven, U.K.). Particle morphology was studied with a transmission electron microscope (JEM1230, Japan). Fourier transform infrared (FT-IR) spectra of the UCNPs were recorded on a Spectrum One (B) spectrometer (AVATAR370, USA).

Synthesis

and

Surface

Modification

of

NaYF4:Yb3+,

Er3+

Upconversion

Nanoparticles (UCNPs). UCNPs were synthesized according to the previously reported procedure.39 Typically, 0.78 mmol YCl3·6H2O, 0.20 mmol YbCl3·6H2O and 0.02 mmol ErCl3·6H2O were added to a 100 mL three-neck round-bottom flask containing 8 mL of OA and 15 mL of 1-ODE, and subsequently heated to 160 °C to form a transparent solution under an atmosphere of nitrogen. The reaction mixture was agitated for 30 min at 160 °C under vacuum, and then subsequently cooled down to room temperature. 10 mL of methanol containing NaOH (0.25 M) and NH4F (0.4 M) was added into the flask and stirred for 30 min. The mixture was heated to 100 °C and stirred for appropriate time in order to 6 ACS Paragon Plus Environment

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remove methanol, and then subsequently heated to 300 °C. After 1 h of reaction, the resultant mixture was cooled down to room temperature. The obtained raw products were precipitated and then washed for several times with ethanol and cyclohexane by centrifugation, and dried at 50 °C for 12 h under vacuum atmosphere. The PAA-functionalized UCNPs were prepared using a modified ligand exchange strategy via the procedure reported by Xia et al.40 Typically, a mixture of PAA (20 mg) and H2O (10 mL) was first heated at 50 °C under vigorous stirring for 30 min. 10 mg of UCNPs-OA was dissolved in a mixed solution of cyclohexane (10 mL) and ethanol (5 mL), and subsequently the UCNPs-OA was added into the PAA solution and stirred for 4 h. After cooling down to room temperature, the lower liquid was collected until the mixture was layered. UCNP-PAA was obtained by centrifugation at 10000 rpm for 10 min. Finally, the product was washed with ethanol twice and redispersed in 10 mL of water.

Synthesis of GNPs by Seed-Mediated Growth. The preparation of homogeneous GNPs was based on the seed-mediated growth method as reported in the previous literature.10,13 In brief, the fabrication procedure contained two parts, including the seed preparation and particle growth process. For the preparation of the seed solution (with diameter ~18 nm), 10 mL of sodium citrate (2.2 mM) and 1.03 mL of HAuCl4·4H2O (24.28 mM) were added into 98.97 mL of H2O and boiled for 20 min at 110 °C under vigorous stirring.



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To generate larger particles, 30 mL of sodium citrate solution (13 mM) was added into 48 mL seed solution (pre-diluted by 8×). The mixture was heated to 120 °C for 10 min, followed by adding 20 mL HAuCl4·4H2O (2.35 mM) gradually. After that, the sample was heating for 10 min and then gradually cooled down to room temperature.

Preparation of Capture Antibody-Modified GNPs and Detection Antibody-Modified UCNPs. To conjugate anti-PSA capture antibody (Ab2) to the surface of GNPs, anti-PSA capture antibody modification solution was prepared first. Briefly, 2 µM NHS-PEG-SH and 2 µM anti-PSA capture antibody were mixed with 50 µL of filtered Tris-buffered saline (10 mM, pH 8.5) with gentle stirring for 3 h. 1 mL of 50 nm GNPs stock solution was centrifuged at 4500 rpm for 10 min to remove the extra chemicals in the solution. The pellet was dispersed in 80 µL of 1 mg/mL BSPP solution and kept for 1 h. After that, 30 µL modification solution including anti-PSA capture antibody was added to the concentrated GNPs solution and left to react for additional 3 h. In addition, thiolated methoxyl-polyethylene glycol (CH3O-PEG-SH, MW: 6000) was added as a blocking agent and the sample was further incubated for 2 h. The excessive reagents in the supernatant were removed by centrifugation. Finally, antibody-conjugated GNPs were suspended in 50 µL of Tris-buffered saline (TBS) and stored at 4 °C prior to use. The zeta-potential was then measured to further ascertain the modification process. For the conjugation, the PAA-functionalized UCNPs were activated by excessive N(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC)/ N-hydroxysuccinimide (NHS) in ratio of 1:1 at room temperature for 30 min. Then, 1.2 µM anti-PSA detection antibodies 8 ACS Paragon Plus Environment

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(Ab1) in 10 mmol/L TBS was mixed with 100 µL of 2.0 mg/mL carboxyl-modified UCNPs solution in the same buffer, and the reaction was continued for 2 h in a reciprocating oscillator. After that, 1% bovine serum albumin (BSA) was added and further incubated for 1 h. The anti-PSA detection antibodies conjugated UCNPs were purified by centrifugation and dissolved in filtered TBS buffer.

Detection of Prostate Specific Antigen Based on the LRET System. The LRET-based immunoassay for PSA was conducted by the following procedures. Typically, various concentrations of target proteins were added into a series of tubes including 5 µL UCNPs-Ab1 suspensions, respectively, followed by incubating for 1 h with slow shaking. Subsequently, 10 µL of GNPs-Ab2 was added into each tube. The samples were incubated for another 1 h. After that, the sample was imaged with the home-built upconversion imaging system for SPE measurements. To demonstrate the potential in serum sample analysis, the standard addition method was applied to detect PSA in serum sample from a health volunteer. Briefly, the antigen standards of 5, 10, 20, 30, and 40 pM were spiked into the crude serum samples, respectively, without any dilution or purification. The immunoassays were performed with corresponding nanoprobes, and all data were analyzed as mentioned above. To explore the clinical application potential, we performed the determination of PSA in a purified donor serum sample with the standard addition approach. The detection process was the same as that in pure buffer solution except that the PSA was diluted by the purified serum. The relative standard deviation (RSD) was then adopted to evaluate the accuracy and precision of the results. 9 ACS Paragon Plus Environment


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RESULTS AND DISCUSSION According to the earlier spectroscopic reports, the GNPs in the size range of 50-90 nm provide the highest efficiency for fluorescence quenching toward the closeby UCNPs at the single-particle level.37,38 On this basis, we chose GNPs of ~50 nm as the quenching agent for its good colloidal stability in biological milieu and excellent quenching capability. The GNPs were synthesized with a seed mediated growth method and the morphology was characterized with transmission electron microscopy (TEM) as shown in Figure1a. The particles displayed good water dispersibility and narrow size distribution (51.1±6.8 nm) which was further confirmed by dynamic light scattering measurements (DLS, 58.8 nm). As the UCNPs were fabricated based on the thermolysis method, the surface of the as-synthesized UCNPs was capped with a layer of oleic acid which is insoluble in water. Ligand exchange was then required for the phase transfer as well as the introduction of functional groups for the subsequent biomolecule conjugation. Herein, a water soluble and biocompatible polymer, polyacrylic acid (PAA) was mixed with the as-synthesized UCNPs in water by ultra-sonication. Owing to the large amount of carboxyl groups on the polymer chain, oleic acid could be readily replaced by PAA. Figure 1b shows the TEM image of the PAA-coated UCNPs. It shows clearly that the particles dispersed well in water with diameters around 42.5±1.1 nm. The DLS measurement (50.7 nm) also confirmed that there was no aggregation after the ligand exchange process. In order to further verify the success of the ligand exchange, FT-IR spectroscopy measurement was performed, Figure 1c. Prior to the ligand exchange process, two peaks were observed at 2854 and 2951 cm-1, which were assigned to the symmetric and 10 ACS Paragon Plus Environment

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asymmetric stretching vibrations of CH2 from oleic acid molecules, respectively. In addition, two peaks associated with the asymmetric and symmetric stretching vibrations of carboxylic group (COO-) can be found at 1547 and 1460 cm-1 respectively. After the surface modification with PAA, the profile of its FT-IR spectrum changed noticeably, where the characteristic peaks corresponding to the oleic acid disappeared. A strong peak appeared at 1718 cm-1, which is ascribed to the symmetrical stretching vibration of the COOH. The successful coating of PAA polymers onto the surface of UCNPs not only guarantees the monodispersibility of the UCNPs in biological fluids, but also provides plenty of functional groups for the further protein conjugation. Subsequently, the UCNPs-COOH was modified with the detection antibody via standard EDC/sulfo-NHS chemistry. As illustrated in Table 1, before the conjugation process, the zeta potential of PAA-coated UCNPs is very negative (-33.1 mV), which is attributed to the dense carboxyl groups on the particle surface. Cross-linking of the detection antibody to the UCNPs partly shields the carboxyl groups, resulting in the increase of zeta potential (19.9 mV). For GNPs, the conjugation of the proteins to the surface is relatively more straightforward. By using a thiol group-functionalized polyethyleneglycol polymer (NHS-PEG-SH), the proteins could be readily coated on the GNPs surface via Au-thiol chemistry. About 25 mV augment in the zeta potential was observed after the conjugation process (from -41.9 to -16.9 mV). As depicted in Figure 1d, it is clear that the absorption energy level from GNPs is largely overlapped with the emitted photon energy from UCNPs. It implies that when the distance between the GNPs and UCNPs is close enough (e.g. less than 10 nm), the emitted photons from the UNCPs could be well quenched due to the LRET process. The 11 ACS Paragon Plus Environment


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immunoreaction between the antibody and antigen will drag these two particles together. The gap distance between them is typically less than 10 nm taking into account of the hydrodynamic radius of the proteins. Therefore, the fluorescent signal would be well quenched once the immunoreaction successfully takes place. As a proof of concept, we firstly immobilized detection antibody-functionalized UCNPs on the glass slide surface in a flow channel. Individual monodispersed green spots of UCNPs were readily observed as shown in Figure 2a. To eliminate the nonspecific adsorption, BSA was adopted to block those non-specific binding sites on the glass slide surface. Solution of target antigens was injected into the chamber and incubated with the antibody-functionalized UCNPs for 60 min. The specific antigenantibody recognition enables the effective capturing of the randomly diffusing antigen by the functionalized UCNPs. In fact, when the capture antibody-functionalized GNPs were introduced into the flow chamber, some of the fluorescent spots gradually disappeared (Figure 2b), which is also confirmed by the spectroscopic measurements in the solution sample (Figure 2c). It is worth emphasizing that no photobleaching or photoblinking took place from those UCNPs. The photostability from individual UCNPs was verified to be quiet stable as supported by Figure 2d-e. Therefore, this antigen-dependent fluorescence quenching process could be ascribed to the immunoreaction between the functionalized UCNPs and GNPs. Next, we explored the detection capability of this single particle immune sensor for the quantitative biomarker assay, e.g. for PSA. As shown in Figure 3, the number of fluorescent particles observed on the glass slide surface gradually decreased as a function


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of the PSA concentration. By measuring the counts of fluorescent spots, a concentrationdependent relationship between the counted particle number and target molecule concentration would be established. Herein, to avoid the artificial error for SPE detection among different batches of experiments, a ratio rather than the absolute counted number was utilized for the quantitative assay. The equation is defined as R=(No-Ni)/No, where No is the counted number of UCNPs in the blank control and Ni is the counted number of UCNPs under different target concentrations. As illustrated in Figure 3b, the dynamic range for PSA assay was determined to be 0-500 pM in Tris buffered saline. A linear relationship was found in the range of 0-60 pM. The detection limit of this LRET-based immunosensor was determined to be 1.0 pM from the statistical analysis, which revealed the excellent performance of the nano-platform in quantifying antigens. It is worth noting that the LOD for PSA detection (i.e., 1.0 pM) here is much lower than that of the cut-off PSA concentration in patients’ serum (>117.6 pM), which manifests that this LRETbased immunosensor would be a promising option for the quantitative detection of PSA for early disease diagnosis. On this basis, the specificity of this immunosensor was next examined. Control experiments were performed to evaluate the specific recognition capability of this sensor. Firstly, GNPs without antibody conjugation was used to replace GNPs-Ab2. The other reaction conditions were maintained the same. As shown in Figure 4, even the concentration of the target molecule increased as high as 500 pM, no obvious change was observed on the counts of fluorescent particles on the glass slide surface, indicating no specific interaction between GNPs and UCNPs-Ab1. Similarly, when the UCNPs-Ab1 was replaced by UCNPs (without antibody conjugation), comparable results were 13 ACS Paragon Plus Environment


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observed, Figure 5. The quantitative particle counting results are shown in Figure 6a. To further explore the selectivity of this nano-immunosensing assay for the target antigen recognition, anti-PSA nanoprobes were mixed with different biologically relevant species including human IgG, HSA, Na+ and K+ under the same reaction conditions. As presented in Figure 6b, none of the tested interfering species caused obvious upconversion fluorescence quenching except the target PSA antigen. Encouraged by the excellent sensitivity as well as selectivity toward target antigen detection in biological buffer, we further explored the capability of this sensor in samples of human serum. The serum sample was obtained from a healthy donor. The PSA were spiked in the purified serum with different concentrations. As illustrated in Figure 7a-f, the counted particles on the fluorescence image declined gradually along with the increase in target antigen concentrations. A comparable linear dynamic range (0-40 pM) as that in TBS buffer was achieved, suggesting that the method can be successfully used for the quantitative detection of target antigen in serum samples. The detection limit was estimated to be 2.3 pM, which is also comparable with that in pure buffer assay. In the standard addition assay, the quenching ratio increased linearly with the increased concentrations of target antigen in the standard samples. As shown in Table 2, a percentage recovery of 96.5-107.0% for the spiked PSA samples was obtained, indicating acceptable accuracy of the proposed detection sensor for PSA in serum samples. These results demonstrated that the biosensor developed in this study can be used as a quantitative and qualitative method for PSA detection in complex biological samples. It is worth to emphasize that not only for PSA detection, this detection strategy can also be


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readily extended to other biomolecule detection in serum sample, e.g., carcinoembryonic antigen, alpha-fetoprotein and so on.9,13

CONCLUSION In this work, we demonstrated a digital sandwich immunoassay for cancer biomarker detection via counting of upconversion particles in a flow channel. In the presence of target antigen, the specific immunoreaction between UCNPs-Ab1 and GNPsAb2 effectively brought the donor and acceptor into close proximity, resulting in decrease in the fluorescence signal as induced by LRET. A dynamic range of 0-500 pM and LOD of 1.0 pM for PSA assay were readily achieved based on the quantitative counting of upconversion fluorescent particles on the glass slide surface. Owing to the excellent selectivity of immunoreaction and upconversion fluorescence from UCNPs, this sensor exhibited high specificity toward the target antigen molecules under complicated biological surrounding, i.e., serum sample. No interference was observed from other proteins such as IgG and HSA. Since the LOD of this strategy is much lower than the PSA concentration in serum from the patients, this direct, simple yet sensitive digital enumeration method would find broad applications for the quantitative and selective detection of cancer biomarkers in clinical applications in the future.

ASSOCIATED CONTENT

AUTHOR INFORMATION Corresponding Author 15 ACS Paragon Plus Environment


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*Email: [email protected], [email protected] Author Contributions All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. All author reviewed the manuscript. ACKNOWLEDGMENT This work was supported by national natural science foundation of China (NSFC, Project no. 21522502 and 21405045).


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FIGURES AND CAPTIONS

Scheme 1. Schematic diagram of the light path for the optical microscopic imaging of UCNPs and the principle of digital immunosorbent assay by single-particle enumeration.



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Figure 1. The microscopic and spectroscopic characterizations of UCNPs and GNPs. a) and b) are the DLS size distributions of GNPs and PAA-functionalized UCNPs in water respectively. The insets are the corresponding TEM images. c) The FT-IR spectra of UCNPs before (red line) and after (black line) PAA modification. d) The fluorescence and extinction spectra of UCNPs (green line) and GNPs (red line) respectively.


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Figure 2. a) The fluorescence image of UCNPs-Ab1 on the glass slide surface. b) The fluorescence image of UCNPs-Ab1 from the same area after the introduction of antigen and GNPs-Ab2. c) Luminescence spectroscopic characterizations of the solution sample (containing UCNPs-Ab1 and GNPs-Ab2) before (red) and after (green) the addition of target antigens. d) Representative single particle fluorescence image of UCNPs with long exposure time. e) The time-dependent fluorescence tracks from the individual particles as noted in c).



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Figure 3. a) Target antigen-dependent fluorescence images of UCNPs on glass slide surface. b) The calibration curve of the immunosorbent enumeration assay. c) The linear range for the quantitative assay.


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Figure 4. The specificity assay of the immunoreaction under different antigen concentrations (0-500 pM). The immunoreaction conditions were the same except that GNPs-Ab2 were replaced by GNPs.



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Figure 5. The specificity assay of the immunoreaction under different antigen concentrations (0-500 pM). The immunoreaction conditions were the same except that UCNPs-Ab1 were replaced by UCNPs.


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Figure 6. a) The counted number of fluorescent particles on the glass slide surface from the control experiments as shown in Figure 4 (Ⅰ) and 5 (Ⅰ) respectively. b) The specificity assay of the immunoassay.



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Figure 7. a)-f) The representative fluorescence images of the immnunoassay in serum sample. g) The calibration curve.


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Table 1. Zeta potential measurements of the particles with different functionalizations.

Name

UCNPs-PAA

UCNPs-PAA-Ab1

GNPs

GNPs-Ab2

Zeta/mV

-33.1

-19.9

-41.9

-16.9

Table 2. The standard addition assays and recovery rate estimation.

Sample

Spiked (pM)

Detected (pM)

Recovery (%)

1

3

3.20±7.02

107

2

15

14.48±2.29

96.5

3

35

34.10±2.83

97.4



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