An Enzyme-Free Immunosorbent Assay of Prostate Specific Antigen

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An Enzyme-Free Immunosorbent Assay of Prostate Specific Antigen Amplified by Releasing pH Indicator Molecules Entrapped in Mesoporous Silica Nanoparticles Fengying Shao, Lianhua Zhang, Lei Jiao, Xiaoying Wang, Luyang Miao, He Li, and Feimeng Zhou Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 19, 2018

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Analytical Chemistry

An Enzyme-Free Immunosorbent Assay of Prostate Specific Antigen Amplified by Releasing pH Indicator Molecules Entrapped in Mesoporous Silica Nanoparticles Fengying Shaoa,1, Lianhua Zhangb,1, Lei Jiaoa, Xiaoying Wangc, Luyang Miaoa, He Lia,*, Feimeng Zhoua,* a

Institute of Surface Analysis and Chemical Biology, University of Jinan, Jinan 250022, China

E-mail: [email protected] (H. Li); [email protected] (F. Zhou) b

Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China

c

College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China

ACS Paragon Plus Environment

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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: An enzyme-free titer plate-based colorimetric assay utilizing functionalized mesoporous silica nanoparticles (MSNs) entrapping pH-indicator molecules has been developed. Pores in the silica nanoparticles were functionalized with phenyltrimethyloxysilane so that pH indicator molecules (thymolphthalein or TP in the present case) can be tightly entrapped through π−π conjugation. To detect prostate specific antigen (PSA), the TP-containing MSNs were coated with polyethylenimine (PEI), which favors the attachment of the positively charged secondary anti-PSA antibody. The entrapped thymolphthalein molecules can be readily released from the pores with a simple addition of alkaline solution. The resultant bifunctional MSNs were used for signal-amplified detection of PSA captured by the primary antibody pre-immobilized in the wells of a plate. Our method possesses a wide dynamic range (0.5 to 8000 pg/mL) wherein the adsorption of the bifunctional MSNs obeys a modified Langmuir isotherm. A detection limit (LOD) down to as low as 0.36 pg/mL can be attained. Owing to the size uniformity of the MSNs and the obviation of enzyme molecules employed in the enzyme-linked immunosorbent assay (ELISA), excellent reproducibility (RSD = 1.12%) was achieved. The selective detection of PSA in human serum samples demonstrates the amenability of our method to detecting important biomarkers in complex biological samples, whereas the performance of the assay in a titer plate ensures high throughputs and obviates the use of expensive instruments. Both of these features are prerequisites for clinical settings wherein a great number of samples need to be analyzed in a timely fashion.


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INTRODUCTION Since 1986, prostate specific antigen (PSA) has been used as a major clinical biomarker for diagnosis of possible prostate cancer.1 Generally, the PSA concentration is below 4 ng/mL in healthy human sera, and a rise of serum PSA above 10 ng/mL is taken as a sign of problems such as prostatitis and urinary tract infection or in severe cases prostate cancer.2,3 Various techniques have been employed to detect PSA. These techniques include, but are not limited to, spectrofluorimetry,4 surface-enhanced Raman scattering,5 electrophoretic mobility assay,6 and electrochemiluminescence (ECL)7. Assays based on these techniques either require relatively expensive or specialized instruments or involve laborious and time consuming. Some may encounter issues such as instability and relatively poor selectivity. Because of its simplicity, sensitivity, high throughput, and amenability for automation, enzyme-linked immunosorbent assay (ELISA) has become a powerful bioanalytical and immunological tool for life science research. 8 ELISA is considered as the “gold standard” for biomarker detection in clinical laboratories, due to its superb sensitivity and capability of measuring simultaneously contents inside many wells on a titer plate.9,10 However, the use of enzymes for traditional ELISA leads to variability and false positives due to possible loss of enzyme activities or changes in enzymatic activities caused by temperature and pH fluctuations in the ambient or analyte solutions. 11 , 12 To circumvent such a problem while retaining the advantage of signal amplification inherent in enzymatic reaction, researchers have endeavored to replace enzymes involved in the conventional ELISA.13-16 Previously, we attached a pH indicator (phenolphthalein), through hydrophobic interaction, along with an antibody that is specific to carcinoembryonic antigen, via covalent binding, onto graphite-phase carbon nitride nanosheets. The resultant nanocomposites were employed in a


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colorimetric assay via addition of an alkaline solution after the formation of the antibody-antigen conjugate. Despite the proof-of-principle experiments demonstrating the new material design and analytical applications,17 the nanosheets have the following limitations. They vary significantly in size and their planar structure does not tightly withhold the indicator molecules. Both issues can affect the assay reproducibility when aliquots of the nanocomposites synthesized in the same or different batches are used in replicate assays or analyses of the same sample at different times. Furthermore, in clinical settings where many samples need to be analyzed, it is desirable to use materials that are cheaper or simpler to make than the graphite-phase carbon nitride nanosheets. In the field of nanomaterials and nanotechnology, efforts have been embarked on the synthesis and functionalization of mesoporous silica nanoparticles (MSNs) to afford them large surface areas, tunable surface functionality, and biocompatibility. The impetus stems from the potential applications of MSNs in areas as diverse as adsorbant production, 18 drug delivery, 19 biomolecular detection20 and theranostics21,22. MSNs are especially suitable for incorporating a large amount of cargo molecules. The types of cargo molecules encapsulated by MSNs include small-molecule drugs,23 peptides,24 and dyes.25 Surprisingly, to the best of our knowledge, incorporating pH indicators and releasing them controllably for bioassays have not been reported. We envision that the aforementioned problems inherent in our previously synthesized nanosheet/indicator composite can be mitigated by entrapping pH indicator molecules in the pores of MSNs. Moreover, MSNs with high uniformity can be straightforwardly and costeffectively synthesized. These features reduce variability of the assays and are more amenable to clinical applications. Herein we report on the synthesis of a new type of bifunctional MSNs that can recognize PSA and generate a follow-up color change. To entrap the pH indicator (thymolphthalein) molecules, MSN poles were first derivatized with phenyltrimethoxysilane


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whose phenyl rings provide π−π interaction with thymolphthalein. Due to the hydrophobic interaction, many more TP molecules can be entrapped into the pores of MSNs to enhance the sensitivity of this method. Furthermore, the phenyl groups adjacent to the top of each MSN pore prevent water molecules from entering into the interior of the pore. As a result, the TP molecules are remain trapped and stable inside the pore. To render specific recognition of PSA, the thymolphthalein-containing MSNs were coated with polyethyleneimine whose positive charges facilitate the electrostatic attachment of the negatively charged secondary anti-PSA antibody. The as-prepared bifunctional nanocomposite was utilized a sandwich assay of PSA wherein the analyte capture and detection can be carried out in different cavities of a 96-well titer plate. Thus, the assay is readily adaptable to any commercial plate readers, allowing simple yet sensitive detections to be performed in a high-throughput fashion. EXPERIMENTAL SECTIONS Chemicals and Materials. PSA and PSA primary (Ab1 ) and secondary(Ab2) antibodies were acquired from Shanghai Linc-bis Science Co. (China), Ltd. Bovine serum albumin (BSA, 9699%) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Phenyltrimethoxysilane (PTTS), tetraethylorthosilicate

(TEOS),

N-cetyltrimethylammonium

bromide

(CTAB,

≥ 99%),

polyetherimide (PEI) and thymolphthalein (TP) were purchased from Sinopharm Chemical Reagent Shanghai Co., Ltd, (China). All other chemical reagents were analytically pure. Phosphate buffered saline (PBS, 0.1 M) were prepared by mixing KH2PO4 with Na2HPO4 stock solutions with pH adjusted using NaOH. Ultrapure water was used throughout the experiments. Polystyrene 96-well plates were obtained from Corning Corporation (New York, USA).


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MSN Synthesis, Silalization, Indicator Entrapment and Antibody Attachment. We modified the MSN synthetic procedure and developed the subsequent silanization step.26 Briefly, 0.50 g of CTAB was dissolved in 240 mL water. Next, sodium hydroxide (1.75 mL, 2 M) was added, followed by adjusting the solution temperature to 80°C. TEOS (2.50 mL) was first introduced dropwise to the surfactant solution. To silanize the TEOS-based MSNs, the above step immediately proceeded to dropwise additions of PTTS. The resultant solution was continually stirred for 2 h to yield a white precipitate. The solid product was separated by centrifugation, and washed with water and ethanol three times. Subsequently, the purified nanoparticles were dried in a vacuum container at 60°C overnight. To remove the surfactant (CTAB), 1.50 g of as-synthesized MSNs were refluxed for 24 h in a mixture of 9.0 mL of HCl (37.4%) and 160 mL of methanol, and the final product was washed extensively with water and methanol. The resultant surfactant-free MSNs were dried in vacuo to remove any solvent inside the pores. Negatively charged OH ﹣ groups can be attached to the MSNs through such a procedure.27 As will be shown in the Results and Discussion section, the inclusion of PTTS renders spatially accessible phenyl moieties to the interior of the MSN pores, facilitating the entrapment of a larger number of indicator molecules. Coating of the MSNs with polyetherimine (PEI) followed a published study.28 Briefly, 500 mg of MSNs were dispersed in 40 mL of water into which 300 mg of PEI was added. The reaction mixture was stirred at 25°C for 12 h. During this process, the positively charged PEI molecules were adsorbed onto the negatively charged MSN, thus producing PEI-coated or -wrapped MSNs (PEI-MSNs). The PEI-MSNs were then centrifuged and washed with methanol and dried in air. To introduce the indicator thymolphthalein (TP) into the pores, 10 mg of PEI-MSNs were


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dispersed in 1 mL of PBS buffer into which 100 µL ethanol solution of 1 mM TP was added. This step was followed by gently shaking the solution for 2 h. The above-prepared TP@PEI-MSNs (1 mg) were dispersed in 1 mL of PBS (pH 7.4). Next, the secondary antibody of PSA (Ab2) was added, and the solution was stirred at 4°C for 6 h. The negatively charged Ab2 molecules were attached to TP@PEI-MSNs via electrostatic interactions. The mixture was centrifuged (at 4297 g for 15 min) to remove unbound Ab2 molecules. The final bifunctional TP@PEI/Ab2-MSNs were dispersed in 1 mL of PBS (pH 7.4) and stored at 4°C. Instruments. Transmission electron microscopic (TEM) images were recorded on a field emission microscope (Philips CM200 UT, USA) and scanning electron microscopic (SEM) images were obtained with a Quanta FEG250 field-emission environmental SEM (FEI, United States) operated at 4 kV. The absorbance values of solutions confined in the wells were recorded by a Safire2 microplate reader (Thermo Fisher Scientific, USA). Fourier transform infrared (FTIR) spectra were collected from an infrared spectrometer (Model 410, JASCO, Japan). Brunauer−Emmett−Teller (BET) surface areas and pore size distribution were measured and calculated by a fully automatic surface area and pore size analyzer (Model ASAP 2020, Micromeritics Instrument Corporation, USA) with temperature set at 77 K. Enzyme-Free Colorimetric Immunoassays and Chemiluminescence Measurements. The Ab1 solution (50 µL, 10 µg/mL) was added into each well of the 96-well PS plate and allowed to stand overnight at 4°C. After decanting the solutions, the plate wells were washed with PBS (pH 7.4, 300 µL, three times) and the well surface was blocked with 5% BSA (50 µL) for 1.5 h at 25°C. This was followed by rinsing with copious PBS (pH 7.4, 300 µL) solution for three times. 50 µL PBS solutions containing various PSA concentrations (0.5, 1, 3, 5, 8, 10, 50, 100, 1000,


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5000, and 8000 pg/mL) were then added to the wells and allowed to stand at room temperature for 1.5 h. Wells containing only PBS served as the blank. Upon washing with PBS three times (300 µL each time), 50 µL TP@PEI/Ab2-MSNs were added into the wells. The plate was covered with a plate sealer and incubated at room temperature on a shaker for 1 h. Any unbounded TP@PEI/Ab2-MSNs were washed away by PBS three times. Finally, into each well 0.1 M NaOH (pH 13.0, 100 µL) was added, and the plate was shaken for 150 s. The absorbance values at 593 nm were recorded. The enzyme-free colorimetric immunoassay was used to measure the serum PSA levels of five healthy donors and five prostate cancer patients. The results were compared to those obtained from chemiluminescence analyses performed at Renji Hospital (Shanghai, China) on a Beckman DXI800 immunoassay system (Brea, CA, USA). RESULTS AND DISCUSSION

Scheme 1. (A) The synthesis and derivatization of TP@PEI/Ab2-MSNs and (B) steps of the enzyme-free immunosorbent assay of PSA using TP@PEI/Ab2-MSNs for amplified colorimetric detection in a 96-well plate (B). The reaction involved in releasing the TP molecules by 0.1 M NaOH is shown at the bottom of panel B.


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Panel A of Scheme 1 illustrates the key steps for synthesizing the TP@PEI/Ab2-MSNs, while panel B shows our enzyme-free colorimetric detection of PSA with signals amplified by these TP@PEI/Ab2-MSNs. TEOS-based MSNs can entrap TP molecules into the mesopores via hydrogen bonding, while π−π stacking rendered by PTTS-silanized MSNs encapsulate additional TP molecules (cf. detailed discussion below). For simplicity, we refer the untreated MSNs as TEOS-based MSNs. In our detection scheme, the primary antibody (Ab1) molecules are immobilized in wells of a microtiter plate and the uncoated sites are blocked with bovine serum albumin (BSA). When the wells that have been exposed to PSA-containing solutions are filled with the TP@PEI/Ab2-MSN solution, PSA molecules captured by Ab1 will be capped by TP@PEI/Ab2-MSNs. The detection is based on the color change accompanying the release of TP molecules via addition of 0.1 M NaOH into the wells. As shown by the reaction at the bottom of panel B, deprotonation of the carboxyl and hydroxyl groups by OH− weakens the interaction between TP2− dianions and the pore surface of MSNs. As a consequence, TP2−released from the pores changes the color of the solution. We chose TP instead of phenolphalein as used in our previous work17 because phenolphthalein has a disadvantage in that the rate of decolorization of red deprotonated phenolphthalein at pH 11–12 is significant. The color will fade after 10 min. In contrast, the blue color of the deprotonated thymolphthalein remains for at least 1 h at pH 12 and higher.


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Figure 1. (A) A TEM image of MSNs; (B) A BET nitrogen sorption isotherm of MSNs; (C) BJH pore size distribution plot; (D) FT-IR spectra of TEOS-based MSNs (black curve) and the PTTS-silanized MSNs (red); (E) Photographs of PEI-MSN, TP@PEI-MSNs, and TP@PEI/Ab2-MSNs before and after exposure to 0.1 M NaOH.

As the key to our assay is the synthesis of the bifunctional TP@PEI/Ab2-MSNs, we characterized these nanoparticles throughout their preparation. The TEM image (Figure 1A) confirmed that the MSNs are indeed spherical and the inset shows their size distribution. The average diameter is about 66 ± 7.58 nm and the size distribution is quite narrow. The N2 gas adsorption and desorption isotherms of these MSNs (Figure 1B) showed little hysteresis with distinct adsorption and desorption steps at an intermediate P/P0 value of about 0.20−0.35. The BET surface area measurement yielded a value of 1077.6 m²/g and an average pore diameter of 2.6 nm was estimated from the BJH curve (Figure 1C). The presence of PTTS on the surface of the PTTS- MSNs was confirmed by FT-IR (Figure 1D). When compared to the spectrum of TEOS-based MSNs (black curve), silanization of MSNs by PTTS (red curve) attenuated the bands characteristic of TEOS. Specifically, the absorption bands at 3440 cm−1 (O−H stretching), 785 cm−1 (C-H out-of-plane stretching), 1073 cm−1 (Si−O scissoring) and 1478 cm−1 (C−H scissoring), were decreased by 51.3−79.7%. This trend is expected as parts of the pores are now


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covered with PTTS molecules. Indeed PTTS-silanized MSNs exhibit the absorption band characteristic of C−H of the phenyl ring at 2900 cm-1. The PEI-MSN, TP@PEI-MSNs, and TP@PEI/Ab2-MSNs solutions are all opaque (cf. Figure 1E), suggesting that the three chemically and/or biochemically modified MSNs are suspended in solution. The latter two changed color rapidly when 0.1 M NaOH solution was added (cf. the bottom panel of Figure 1E). This observation indicates that TP molecules were indeed entrapped into the pores and their deprotonation cause them to egress out of the pores.

Figure 2. (A) Absorbance at 593 nm vs. TP concentrations from 0.01 to 8 mM, along with a photograph of the wells containing these solutions. (B) Absorbance at 593 nm vs. [TP] added into TEOS-based MSNs (curve b) and the TPPS-treated MSNs (curve a). (Inset: Photographs of two rows of a titer plate showing the color changes caused by the different amounts of TP released from PEI-coated TEOS-based MSNs (row 2) and PEIcoated PTPS-treated MSNs (row 1)). In all solutions, the MSNs were kept at 0.5 mg/mL.

We resort to UV-vis spectrometry to determine the amounts of TP entrapped by PEI-coated TEOS-based MSNs and PTTS-treated MSNs. As shown in Figure 2A and its inset, the absorbance at 593 nm is proportional to [TP] from 1 to 100 µM, suggesting that the TP amount entrapped in MSNs can be quantified if they can be released into a solution. The photographs in Figure 2B contrasts the effect of silanization of MSNs on the amount of TP entrapped. MSNs, owing to the presence of silicate structure, can incorporate TP (cf. structure depicted in Scheme 1)


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via hydrogen bonding.29 With the addition of NaOH, the neutral TP molecule becomes a highly charged dianion (cf. reaction in Scheme 1) and the hydrophilic interaction overcomes the hydrogen bonding to release the blue-colored TP2− from the pores into the solution (row 2 inset Figure 2B and black curve of Figure 2B). It is obvious that the color changes are more intense in row 1 inset Figure 2B and the absorbance values of the red curve in Figure 2B are greater. This is because the phenyl rings on the PTTS-treated MSNs, through π−π stacking, have helped incorporate additional TP molecules into the MSN pores. The maximum amounts of TP entrapped by TEOS-based MSNs and PTTS- treated MSNs were determined to be 9.0 and 19.0 mg/g, respectively. For both types of nanoparticles, the amounts of TP entrapment increase with [TP] in the solutions and reached saturation at ~5 mM.

Figure 3. (A) Photographs of TP@PEI/Ab2-MSNs solutions with pH adjusted from 5.0 to 13.5. Absorbance of TP@PEI/Ab2-MSNs plotted against the time (B) and different temperatures (C) after adding 0.1 M NaOH. The photographs above the two panels show the color intensities of the solutions. In all cases, 1 mg/mL of TP@PEI/Ab2-MSNs was used.


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We next assessed experimental variables to attain the best assay performance. Shown in Figure 3A are juxtaposed centrifuge tubes containing 1 mg/mL TP@PEI/Ab2-MSNs solutions whose pH values were adjusted from 5.0 to 13.5. It can be seen that at pH 9.0 the color has turned into pale blue, consistent with the transition range of TP (9.3−10.5)30. However, the color is most intense at pH ≥ 12.0. We therefore used 0.1 M NaOH in our assays (vide infra) to release TP from the TP@PEI/Ab2-MSNs. We also examined the minimum time required for completely releasing TP. From the absorbance-time plot in Figure 3B, it is apparent that 180 s is sufficient for completely deprotonating and releasing the entrapped TP molecules. Finally, to ensure that temperature fluctuations do not change the color (absorbance) intensities of TP molecules released from the TP@PEI/Ab2-MSNs, we incubated solutions containing 0.1 M NaOH and TP@PEI/Ab2-MSNs from 20 to 30 oC. That the absorbance value remains essentially unchanged indicates the TP deprotonation and release are independent on the temperature variation in this range.

Figure 4. Absorbance values of TP@PEI/Ab2-MSN solutions at 593 nm plotted against different PSA concentrations, along with the curve fitting based on the Langmuir isotherm (red curve). The inset shows the linear portion and its linear regression (red line). Error bars are RSD (n =5).


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With the experimental parameters optimized, we explored the use of TP@PEI/Ab2-MSNs for sensitive immunoassay of PSA. As shown in Figure 4, the absorbance values of TP released from TP@PEI/Ab2-MSNs capping different amounts of PSA increase sharply with the PSA concentration from 0.5 pg/mL to 1 ng/mL. The range spans over three orders of magnitude, and the signal begins to level off beyond 1 ng/mL. The data can be well fitted with the Langmuir isotherm (red curve). The linear portion of the curve spans over 2 orders of magnitude (red line in Figure 4). The relative standard deviations (RSD), shown as the error bars in Figure 4, are all below 1.2%, indicating excellent reproducibility of our method. We attributed the reproducibility to the size uniformity of the TP@PEI/Ab2-MSNs and the tight TP entrapment. To evaluate the stability of the TP@PEI/Ab2-MSNs, we stored them at 4°C and used them for the same assay 2 and 4 days after the initial measurement. The PSA concentration determined was 97.2% and 93.5% of the originally determined value, respectively. The stability TP@PEI/Ab2-MSNs is much greater than indicator molecules (phenolphthalein or PP) attached to carboxylic functionalized planar graphite-phase carbon nitride (cC3N4) nanosheets reported by us.17 As contrasted in

Figure 5. The percentages of phenolphthalein lost from PP-covered Ab2-cC3N4 nanosheets (black squares) and thymolphthalein from TP@PEI/Ab2-MSNs (red dots) over time.


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Figure 5, after being suspended in water, about 10% of PP adsorbed onto the PP-covered Ab2cC3N4 nanosheets (PP-Ab2-cC3N4) are lost in about 3.5 h and the loss continued thereafter (black squares). In contrast, only 3% of TP molecules were lost from TP@PEI/Ab2-MSNs (red dots) and the loss ceased after 2 h. The good stability of our TP@PEI/Ab2-MSNs is an attractive feature when compared to the enzyme-labeled antibody, as the enzymatic activities can be lost or degraded quickly over time. Table 1. Comparison of different methods for PSA assays Method

Linear Range

LOD

Reference

Electrochemical impedance

0.1−100 ng/mL

1.0 pg/mL

[31]

Reverse colorimetric immunoassay

0.05−20 ng/mL

30 pg/mL

[32]

Photoelectrochemical

0.01−100 ng/mL

2.7 pg/mL

[33]

Fluorescence

0.1−100 ng/mL

27 pg/mL

[34]

Electrogenerated chemiluminescence

0.01−8 ng/mL

8 pg/mL

[35]

Electrochemical immunosensor

0.05−50 ng/mL

15 pg/mL

[36]

ELISA

0.001−0.064 ng/mL

0.76 pg/mL

[37]

This method

0.0005−0.01 ng/mL

0.36 pg/mL

This work

The detection limit (LOD), estimated using the slope deduced from the inset of Figure 4 and the difference in the standard deviations between 0.5 pg/mL of PSA and the blank, is 0.36 pg/mL. Such a LOD compares favorably with those obtained with other techniques, some of which require relatively expensive or specialized instruments31-37 (Table 1). Particularly worth noting is that our LOD compares favorably to that achievable with the conventional ELISA. This


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suggests that the signal amplification by releasing multiple TP molecules entrapped in each MSN can rival that inherent in enzymatic reactions. Table 2. PSA Concentrations in Sera Measured by Chemiluminescence and This Method Method

Detection (ng/mL)

Chemiluminescence

0.52 1.27 1.87 2.62 3.55 4.27 5.21 6.24 6.81 7.74

Immunosorbent assay with TP@PEI/Ab2-MSNs

0.54 1.24 1.85 2.57 3.68 4.19 5.20 6.29 6.62 7.63

(ng/mL)

0.029

Sd

0.070

tcalculated

1.31

̅ : the mean value of the differences. Sd: standard deviation of the differences. The tabulated student’s t value at 95 % confidence is 2.26.

To demonstrate the viability of our method for real sample analysis, we measured the PSA levels in sera samples of ten donors (five healthy donors and five with prostate cancer patients) and compared the results to those obtained with chemiluminescence method. All the serum samples were obtained from the Ren Ji Biobank, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, with written consent from all patients. The extracted blood was allowed to stand at room temperature for 30 min. Afterwards, the serum samples were separated by centrifugation at 605 g for 10 min and then assayed. If the specimens are stored, serum can be aspirated and stored in the tube at −80°C until analysis. As can be seen from Table 2, results from the two methods both indicate that the PSA levels in the cancer patients have exceeded the threshold value (4 ng/mL). We performed a student’s t test, which revealed that at 95 % confidence level, the differences in the PSA values between our method and that measured using chemiluminescence in the hospital are statistically insignificant (tcalculated < ttable).38 This is expected as both methods use the same primary and secondary antibodies, and the only but key


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difference is the replacement of the chemiluminescence readout with a simple yet effective colorimetric detection. Thus, our method is accurate and should be amenable for assays of cancer biomarkers in clinical settings.

CONCLUSION We have developed a new type of bifunctional MSNs and extended them for enzyme-free immunosorbent assays. The biomolecular recognition is realized by antibody molecules electrostatically adsorbed onto the MSNs (the antigen used in our proof-of-concept experiment is prostate-specific antigen), whereas colorimetric detection can be accomplished by releasing the reporter molecule (thymolphthalein, a pH-sensitive indicator) entrapped in the MSN pores into the analyte solution. Silanization of silicate-based MSNs with phenyltrimethoxysilane provides π−π stacking for TP, allowing additional TP molecules to be entrapped in the pores of MSNs. As each MSN can incorporate a large number of reporter molecules, signal amplification is attained, allowing the resultant assay to yield a detection limit (0.36 pg/mL) that is superior to other existing methods that require more expensive and specialized instruments. The amenability of our method for real sample analysis demonstrates that it can rival conventional ELISA in clinical laboratories in terms of its accuracy, sensitivity, and throughput. The obviation of enzyme in our assay cuts down the cost of the assay and avoids the issues in conventional ELISA, such as enzyme denaturation and variation of enzymatic activities with temperature. Owing to the stability and robustness of the bifunctional TP@PEI/Ab2-MSNs, our method is highly reproducible. The generality of our approach also allows other biomarkers to be detected so long as their secondary antibodies can be electrostatically attached to the TP-encapsulating MSNs.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (H. Li). * E-mail: [email protected]; [email protected] (F. Zhou). ORCID He Li: 0000-0003-3462-1377 Feimeng Zhou: 0000-0002-2568-765X Author Contributions H.L. and F.Z. designed and directed this study. F.S., L.Z., J. L., X. W., and L. M. conducted the experiments and data collection. F.S. and L.Z. contributed equally to this work.

ACKNOWLEDGMENT The authors would like to thank the financial support from the Natural Science Foundation of China (No. 21245007 and 81000976), the Natural Science Foundation of Shandong Province (No. ZR2017MB017). F. Zhou and X. W. also thank support of a grant from the National Key Basic Research Program of China (2014CB744502), and a 2011 Collaborative and Innovative Grant from Hunan Province of China.

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