CN111735869A

CN111735869A - Protein detection reagent and detection method

Info

Publication number: CN111735869A
Application number: CN202010477365.9A
Authority: CN
Inventors: 陈若虹; 陈保卫; 栾天罡; 刘洪涛
Original assignee: National Sun Yat Sen University
Current assignee: Sun Yat Sen University; National Sun Yat Sen University
Priority date: 2020-05-29
Filing date: 2020-05-29
Publication date: 2020-10-02

Abstract

The application discloses a detection reagent and a detection method for protein. The detection reagent and the established detection method have the double effects of preconcentration and signal amplification, have good specificity and repeatability, have wide linear range and high sensitivity, and can be used for clinical analysis.

Description

Protein detection reagent and detection method

Technical Field

The application belongs to the field of analysis and detection, and particularly relates to a detection reagent and a detection method for protein.

Background

In recent years, the incidence of tumors has been on the rise in the elderly and in young adults. However, conventional clinical approaches do not meet the urgent need for rapid and accurate diagnosis of tumors and related clinical studies. Several biological markers have been reported for clinical screening of tumors. Thus, early diagnosis of tumors is made possible by rapid, high-throughput and patient-friendly assays that combine both quantitative and specific assays to determine biological markers.

Before the instrument detects, an efficient and selective preconcentration technology is needed to detect the target object sensitively. Membrane-based technologies are used for electrophoresis, electrochemistry, and microfluidics due to their high efficiency in protein enrichment. The target protein is attached to the membrane mainly by non-specific adsorption, which depends to a large extent on the Molecular Weight (MWs) and the ionic charge of the membrane components. Therefore, improving the selectivity of the membrane to protein antigens remains one of the pressing problems in the art.

Although a variety of analytical detection methods have been reported for the analysis of targets such as drugs, proteins, metabolites and bacteria in biological samples, accurate determination of low levels of targets to complex substrates in biological samples remains a significant challenge.

Disclosure of Invention

In one aspect, the present application provides a reaction platform comprising a support carrying directly or indirectly a capture element.

In some embodiments, the support and capture element further carry an enrichment element therebetween. The enrichment element can be one or more, and can also refer to a group/set of reagents or components which are matched with each other to have enrichment effect. "enrichment" can refer to either primary or multiple/secondary enrichment.

In some embodiments, the enrichment element is selected from at least one of biotin, streptavidin, and avidin.

In some embodiments, the enrichment element is selected from at least one of biotin, streptavidin.

In some embodiments, the enrichment element is selected from the group consisting of biotin and streptavidin.

In some embodiments, the capture element is selected from at least one of an antibody or an aptamer.

Capture elements include those reagents or components that can capture a substance of interest (e.g., an antigen). In some embodiments, such capturing comprises specific capturing.

In some embodiments, the capture element on the reaction platform comprises an antibody.

In some embodiments, the support bears, or is modified to bear, at least one of the following groups: amino, carboxyl, aldehyde, epoxy; preferably, the support bears, or is modified to bear, an amino group.

In some embodiments, the support is selected from at least one of nylon, static electricity proof silk, cellulose paper, glass, chip, aluminum foil, stainless steel sheet.

In some embodiments, the support is selected from nylon.

In some embodiments, the nylon is selected from Biodyne B nylon. In the prior art, Biodyne B nylon is generally used for enriching nucleic acid because the Biodyne B nylon is charged by itself. In one embodiment of the present application, the inventors surprisingly found that Biodyne B nylon can be used as one of the choices of the support of the reaction platform of the present application, and the reaction platform constructed by using the Biodyne B nylon as a carrier can realize specific capture and/or enrichment of proteins (such as prostate specific antigen) by the functional modification of Biodyne B nylon, thereby overcoming the nonspecific problem of the existing membrane-based technology for target protein adsorption.

In some embodiments, a functionalized reaction platform is prepared with an enrichment element and a capture element that can achieve both specific capture and enrichment of a target protein (e.g., prostate specific antigen) in a test sample.

In one aspect, the present application provides a probe comprising a metal nanoparticle having a mass tag and a capture element attached thereto.

In some embodiments, detection of the concentration of the target protein in the sample is achieved by detection of the signal intensity of the mass tag.

In some embodiments, the mass label is selected from compounds having a thiol group. In some embodiments, the mass tag is linked to the metal nanoparticle by a "metal-S" bond.

In some embodiments, the mass tag is selected from at least one of trimethylolpropane tris (3-mercaptopropionate), N- (2-mercaptopropionyl) glycine, sodium 2-mercaptoethanesulfonate, sodium 3-mercapto-1-propanesulfonate, 4-mercaptophenylboronic acid, 3-mercapto-3-methyl-1-butanol, 4-mercaptophenol, 3-mercaptobenzoic acid, isooctyl 3-mercaptopropionate, butyl 3-mercaptopropionate, isooctyl thioglycolate, 6-mercaptopyridine-3-carboxylic acid.

In some embodiments, the mass label is selected from trimethylolpropane tris (3-mercaptopropionate).

In some embodiments, the capture element on the probe is selected from at least one of an antibody or an aptamer.

In some embodiments, the capture element on the probe is selected from an aptamer.

In some embodiments, the sequence of the aptamer is: 5' -SH- (CH)₂)6-TTT ATT AAA GCT CGCCAT CAA ATA GCT TT-3'。

In some embodiments, the metal nanoparticles are selected from at least one of gold nanoparticles, silver nanoparticles, or platinum nanoparticles.

In some embodiments, the metal nanoparticles are selected from gold nanoparticles.

In some embodiments, the metal nanoparticles are 20-50nm in size; more preferably 30 nm.

In some embodiments, the ratio of the number of metal nanoparticles to the concentration of mass labels, capture elements is (1.8-2.5) × 10¹¹1 (1.25-250) and preferably in a ratio of 2 × 10¹¹：200:1。

In one aspect, the present application also provides a protein detection kit or reagent, including the reaction platform, and/or the probe. In some embodiments, the sample is analyzed by mass spectrometry after being detected by the kit or reagent.

The application also provides a protein detection method, which comprises the following steps:

s1, placing the reaction platform in a sample to be tested, and reacting for 1-2 hours at room temperature;

s2, after the step S1 is finished, mixing the probe with the mixture, and continuously reacting for 1-2 hours;

s3, directly carrying out mass spectrometry on the reaction platform prepared in the step S2.

In some embodiments, the sample to be tested treated by the above method is specifically enriched for the target protein in the sample on the reaction platform, and the mass label is carried by the specific binding of the capture element on the probe, so that the reaction platform can be directly subjected to mass spectrometric detection and analysis. The whole reaction platform is directly subjected to sample introduction and detection, the original state of pretreatment of a sample to be detected is reserved, the possibility of introducing other interference factors is avoided, the specificity is achieved, and the sensitivity of the detection method is further improved by the enrichment effect.

In some embodiments, the mass spectrum is selected from the group consisting of mass open spectrum (AMS).

In some embodiments, the open mass spectrometry is selected from paper spray open mass spectrometry.

In some embodiments, the spray solvent of the paper spray open mass spectrometry comprises at least one of hydrogen peroxide, citric acid, or DL-dithiothreitol.

In some embodiments, the spray solvent comprises DL-dithiothreitol.

In some embodiments, the spray solvent is applied by direct dropwise addition.

In some embodiments, the spray solvent further comprises an alkali metal salt.

In some embodiments, the alkali metal salt is selected from NH₄OH; preferably, NH₄The concentration of OH is 0.1-3M; more preferably 2M.

In some embodiments, the method further comprises the steps of preparing a reaction platform and a probe;

in some embodiments, the preparation of the reaction platform comprises the steps of:

1) contacting the support with an enrichment element to obtain a support loaded with the enrichment element;

2) contacting the support prepared in step 1) with a capture element to obtain a support carrying both an enrichment element and a capture element;

3) incubating the support prepared in step 3) in a blocking agent.

In some embodiments, the support is selected from a shape having at least one corner; preferably, the angle of the angle is selected from 20 ° -150 °; preferably, the angle of the angle is selected from 50 ° -120 °; more preferably 60. The support may be of the above-described shape itself, or may be of the above-described shape obtained by processing.

In some embodiments, the blocking agent is selected from at least one of bovine serum albumin, fatty acid free bovine serum albumin, or skimmed milk powder. The addition of a blocking agent at the end of the reaction platform preparation to reduce non-specific adsorption is important to avoid non-specific interaction between the probe and the reaction platform.

In some embodiments, the blocking agent is selected from fatty acid-free bovine serum albumin.

In some embodiments, the preparation of the probe comprises the steps of:

a) activating the capture element;

b) attaching a capture element to the metal nanoparticle;

c) attaching a mass tag to the metal nanoparticles prepared in step b).

In some embodiments, the capture element in the probe is an aptamer. Aptamers are oligonucleotide sequences that require the first opening of the S-S bond in the duplex for capture, which requires an activator for activating their function.

In some embodiments, the capture element is activated with an activator in step a).

In some embodiments, the activator is selected from tris (2-carboxyethyl) hydrophosphate (TCEP).

In some embodiments, the activator activates the capture reagent for a period of time ranging from 1 to 30 minutes; preferably 5 minutes.

In one embodiment of the invention, the activation time is unexpectedly shortened when TCEP is used to activate the aptamer. The prior art mostly takes 1 hour as the activation time of the TCEP activation aptamer. The inventors have surprisingly found that at this activation time, the gold nanoparticles turn purple and aggregate, while shorter activation times (e.g., below 30 minutes) are effective in avoiding aggregation of the gold nanoparticles. Aggregation of the metal nanoparticles will lead to failure of the probe, while failure of the probe preparation is declared. The probes of the present application require only a short time (within 30 minutes) to complete the aptamer activation. This is relevant to the unique probe design of the present invention.

The application also provides the application of the reaction platform, the probe, the reagent or the kit, or the detection method in protein detection.

In some embodiments, the sample against which the detection is directed is selected from at least one of blood, urine, interstitial fluid, lymph fluid, cerebrospinal fluid, aqueous humor.

In some embodiments, the blood comprises serum, plasma.

In some embodiments, the protein is selected from at least one of an amino acid, a polypeptide, a glycoprotein.

In some embodiments, the protein is selected from at least one of prostate specific antigen, carcinoembryonic antigen (CEA), glycoantigen 125(CA125), alpha fetoprotein, recombinant human neuron specific enolase, glycoantigen 153(CA153), squamous cell carcinoma antigen, P53 protein, human epididymis protein 4.

In some embodiments, the protein is selected from prostate specific antigens.

In one specific example, the detection limit for prostate specific antigen detection can be as low as 4.0pg mL by specific enrichment on the reaction platform, signal amplification of mass tags on the probe, and detection of prostate specific antigen^-1While at the same time 10pg mL^-1To 50ngmL^-1Has good linear correlation in a wide concentration range. In clinical diagnosis, if the concentration of PSA in serum is greater than 4ngmL^-1The patient may be at risk for prostate cancer. The detection limit of this example is 3 orders of magnitude lower than the threshold line for clinical diagnosis, indicating that the method can be used in clinical cases to measure trace protein biomarkersAnzhi, which guides early diagnosis of cancer.

Drawings

Figure 1 is a schematic representation of a membrane reaction platform in series with PSI-MS in one embodiment of the present application. (a) Synthesizing a biotin-streptavidin scaffold with a pre-enrichment function on a membrane; (b) preparing an Apt-AuNPs-TPTM probe; (c) immunoassay and MS detection of PSA. DC: direct current; MS inlet: a mass spectrometer sample inlet; IS: internal standard;

FIG. 2 is a schematic illustration of a method for preparing a reaction platform according to an embodiment of the present disclosure;

fig. 3 is a graph of the results of evaluation of PSA enrichment effect of a reaction platform (i.e., nylon membrane) prepared according to one embodiment of the present application, wherein (a) ECL results for membranes of different treatment methods; (b) comparing the MS signal intensity of the mass labels on the membrane; (c-d) MS spectra of analytes released by the functionalized membrane (c) and the bare membrane (d);

FIG. 4 is a graph of the effect of different PBRs on PSA analysis in one embodiment of the present application;

FIG. 5 is a graph of the effect of concentration of different mass labels on the intensity of mass spectral signals in one embodiment of the present application;

FIG. 6 is a graph of the effect of different TCEP and aptamer reaction times on the intensity of mass spectral signals in one embodiment of the present application;

FIG. 7 is a representation of a probe prepared according to one embodiment of the present application. (a) TEM image of bare gold nanoparticles; (b) TEM images of aptamer-modified AuNPs; (c) UV-Vis spectra of aptamer-modified AuNPs;

fig. 8 is a graph illustrating the effect of different processing regimes on dissociation of a mass tag from AuNP in one embodiment of the present application;

FIG. 9 IS a TPTM and IS mass spectra using high resolution MS in one embodiment of the present application;

figure 10 is a graph of the results of optimizing the spray solvent and desorption performance of PSI-MS in one embodiment of the present application. (a) NH in spray solvent₄Influence of OH concentration on MS signal output; (b) MS signal mode with direct DTT addition: the nylon membrane was clamped to a frame and 5. mu.L of spray solvent (0.1M DTT, 2M NH) was added₄OH, 1mM IS in Acetonitrile (ACN) and directIonizing; (c) MS signal pattern of DTT pre-applied to nylon membrane: mu.L of an equivalent amount of DTT was previously dropped on a nylon membrane, allowed to dry at room temperature, and then the nylon membrane was sandwiched between racks and 5. mu.L of a spray solvent (2M NH)₄OH and 1mM IS in ACN);

FIG. 11 shows the results of specificity and cross-reactivity tests for PSA detection in one embodiment of the present application;

FIG. 12 is a calibration curve for PSA in PBST in one embodiment of the present application; by analyzing the sample containing 50ng mL^-1PBST of PSA obtains the insertion MS spectrogram of PSA; the concentration of PSA in PBST is 0.01, 0.1, 1, 5, 10, 50ng mL^-1；

Fig. 13 is a result of evaluation of reproducibility and stability of a nylon membrane prepared according to an example of the present application.

Detailed Description

The technical solutions of the present application are further illustrated by the following specific examples, which do not represent a limitation to the scope of the present application. Insubstantial modifications and adaptations of the concepts taught herein by others are intended to be covered by the present disclosure.

"comprising" or "including" is intended to mean that the compositions (e.g., media) and methods include the recited elements, but not excluding others. When used in defining compositions and methods, "consisting essentially of … …" is meant to exclude other elements having any significance to the combination of the stated objects. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed application. "consisting of … …" refers to trace elements and substantial process steps excluding other components. Embodiments defined by each of these transition terms are within the scope of the present application.

In this application, "sample" is the same as "specimen".

Reagent and apparatus

Nylon membranes (Biodyne B) were obtained from Poll Life sciences Inc. (Washington harbor, N.Y.; U.S.A.) and had an average diameter of 31.1nm (2 × 10)¹¹Each granulePer mL of granule^-1) Gold nanoparticles (AuNPs) of (a) were purchased from BBI Solutions (kadif, uk). Bovine Serum Albumin (BSA) and fatty acid free BSA were purchased from Sigma-Aldrich (St. Louis, Mo.). Streptavidin (SA), biotin (biotin), N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDCI), DL-Dithiothreitol (DTT), hydrogen peroxide, NH₄OH, citric acid, NaOH, K₂CO₃，NaCl，Na₂HPO₄，KH₂PO₄Acetonitrile, Tris (hydroxymethyl) aminomethane (Tris), ethylene bistrifluoroethylacetic acid (EDTA), ECL luminogenic reagent and Tris (2-carboxyethyl) hydrophosphate (TCEP, ≧ 98%) were purchased from Biotech (Shanghai, China). Trimethylolpropane tris (3-mercaptopropionate) and pentaerythritol tetrakis (3-mercaptopropionate) were purchased from carbofuran (beijing, china). Prostate Specific Antigen (PSA) and biotinylated anti-PSA mouse monoclonal antibody, as well as carcinoembryonic antigen (CEA) and carbohydrate chain antigen 125(CA125) were purchased from Linc-Bio Science (Shanghai, China). Thiol-modified PSA aptamers were synthesized by the manufacturer. The aptamer (Apt) was confirmed by mass spectrometry and its sequence was as follows: 5' -SH- (CH)₂)₆-TTT ATT AAA GCT CGC CAT CAA ATA GCT TT-3'. Phosphate buffered solution (PBS, pH 7.4) was purchased from Gibco (Waltham, UK) and phosphate Tween buffered solution (PBST, pH 7.4) was obtained by adding 0.1% Tween-20 to PBS. The annealing buffer used in this application was as follows: 10mM Tris, 50nM NaCl and 1mM EDTA in deionized water at pH 7.5-8.0. Deionized water (18M Ω cm) was purified using a Milli-QA10 purification system from Millipore (Bedford, Mass.) in America.

Transmission Electron Microscope (TEM) images were performed on a Tecnai G2 Spirit electron microscope (FEI corporation) by dispersing the samples on a copper mesh at 120 kV. UV-Vis absorption spectra were collected at 1. mu.L on a Nanodrop spectrophotometer (Thermo, USA).

Example 1 preparation of the reaction platform

First, the nylon film was cut into an isosceles triangle shape with an internal base of 5mm and a height of 10mm by a custom steel die. Subsequently, the triangular membrane was rinsed 3 times in 0.1M NaOH and deionized water, respectively. Then, the surface is treatedwith-NH₂The film of residues contained 15mg L^-1Freshly prepared EDCI solution of biotin (150mg L)^-1) Gently shaken for 20 minutes to fix the biotin to the membrane.

Thereafter, the biotin-labeled membrane was washed twice with deionized water and once with PBST, and with streptavidin (10mg L)^-1PBST solution) was incubated for 30 minutes with gentle shaking. After three washes with PBST to remove excess streptavidin, the membrane was incubated with biotinylated anti-PSA mouse monoclonal antibody for 30 minutes at Room Temperature (RT).

Finally, the membrane was washed 3 times with PBST and then incubated with blocking agent in PBST for 1 hour at room temperature to reduce non-specific adsorption. After removal of residual blocking agent, antibody-embedded membranes were stored in PBST at 4 ℃ for further use.

EXAMPLE 2 preparation of Probe

The aptamer (Apt) and the mass label with-SH group are fixed on AuNPs in series through Au-S bonds. Since the synthesized oligomer is a single-stranded DNA, a spatial structure that can specifically recognize PSA can be formed by annealing. Thiol-modified linear aptamers were dissolved in up to 100 μ M annealing buffer and incubated at 94 ℃ for 2 min for initial denaturation, followed by slow cooling to room temperature to form the spatial structure.

First, the aptamer was reduced using freshly prepared TCEP (10mM) for 5 minutes to break the S-S bond, and then gently mixed overnight at room temperature to form Au-S bonds to immobilize it on AuNPs. Subsequently, PBS was gradually added to the aptamer-modified AuNP over 2 hours until the total NaCl concentration reached 0.05M, and then the mixture was aged at room temperature for 36 hours. The mass tag trimethylolpropane tris (3-mercaptopropionate) (TPTM) was then added to the above mixture gently mixed for 12 hours to form Apt-TPTM labeled AuNP. Blocking agent BSA was added to a final concentration of 1% (m v)^-1) And incubated for 1h to block unbound binding sites on the surfaces of AuNPs. Thereafter, the mixture was centrifuged at 9000g for 15 minutes at 4 ℃ to remove excess reagent. Finally, the centrifuged material, i.e., the prepared Apt-AuNPs-TPTM probe, was dissolved in PB containing 0.5% BSAST, and stored at 4 ℃. The pH of the mixture was maintained in the range of 7-8 throughout the process to avoid agglomeration of the aunps.

Example 3 immunoreaction of PSA on a reaction platform

Antibody-embedded membranes prepared in example 1 were incubated in PBST containing a series of PSAs for 1 hour at room temperature with shaking, then washed 3 times with PBST. Thereafter, the membrane substrate bound with PSA was incubated with the detection probe prepared in example 2 to form a sandwich immunoassay for 1 h. Finally, the reacted membrane was washed 3 times with PBST and then placed in deionized water for PSI-MS determination.

Example 4 direct PSI-MS

Mass spectra were acquired on an Orbitrap Elite mass spectrometer (Thermo fisher scientific, San Jose, CA) by either positive or negative ion detection mode. Accurate mass measurements were obtained at a mass resolution of 60,000 and mass to charge ratios (m/z) in the range of 200-800 were recorded. Xcalibur 2.2 software (Thermo Fisher Scientific, usa) was used for experimental control and data acquisition.

The mass spectrum confirmation information of the mass label TPTM (trimethylolpropane tris (3-mercaptopropionate)) and the internal standard IS (pentaerythritol tetrakis (3-mercaptopropionate)) IS shown in table 1.

TABLE 1

After completion of the immunoreaction on the reaction platform, the nylon membrane was placed within a distance of about 5mm with its tip pointing towards the MS inlet. Thereafter, a high voltage of positive ionization of 3kV or negative ionization of 2.3kV was applied to the film through a metal clip. Subsequently, 5. mu.L of the solution containing 2MNH₄OH, 1mM Internal Standard (IS) and 0.1M spray solvent of reducing/oxidizing/acidic reagent were added to the membrane. The mass label and IS desorb from the membrane and subsequently ionizes into the MS inlet.

Example 5MS Signal and quantitation

Quantification was performed using MS high resolution scan mode to distinguish the target signal from the matrix signal. Pentaerythritol tetrakis (3-mercaptopropionate), an analogue of TPTM, was used as ISThe concentration of TPTM was calculated. Quantification involving Response Factors (RF) is according to Deng et al^[1]And (4) calculating. RF ═ C_TPTM/C_IS)/(I_TPTM/I_IS) In which C is_TPTMIs the TMTP concentration, C, previously measured by MS_ISIS added to the spray solvent, I_TPTMAnd I_ISMS single intensity for TPTM and IS. The concentration of the mass label when analyzing the sample was calculated as C_{Mass label}＝(I_{Mass label}/I_IS)×C_IS× RF, wherein I_{Mass label}Is the MS intensity of the TPTM transmitted from the sample.

EXAMPLE 6 clinical sample analysis

Human serum samples (used as control serum) from two prostate cancer patients and several healthy people were obtained from women's hospital, Guangdong province, according to the regulations of the local ethical Committee and were cryopreserved at-80 ℃ until use. Before use, the samples were thawed on ice. Spiked samples were prepared by adding PSA to control serum samples by dilution with PBST. The actual samples used to evaluate the results were analyzed by the Chemiluminescent Microparticle Immunoassay (CMIA) method on an ARCHITECT i2000SR immunoassay analyzer (Abbott, usa). PSA quality control samples (7K70-10) were used to ensure the accuracy of PSA measurements.

The results are shown in Table 2.

TABLE 2 analysis results of actual human serum samples at different dilutions

Example 7 Synthesis of Biotin-streptavidin scaffolds and their Effect on PSA preconcentration

On-membrane assays were performed with Electrochemiluminescence (ECL) intensity as the signal output to determine the formation of biotin-SA scaffold on the membrane.

After fixation with biotin, the membrane was embedded with SA, and then reacted with an excess of HRP-labeled biotin to develop color. The darker the color indicates the greater amount of HRP-labeled biotin was embedded, which also indirectly reflects the level of biotinylated PSA antibody on the established membrane platform. FIG. 3a shows that the membrane treated with biotin and streptavidin (D) is darker in color than the controls (A, B and C). The results indicate that the biotin-streptavidin scaffold was successfully synthesized and attached to the membrane, which lays a key foundation for subsequent PSA preconcentration and immunoassay.

The enrichment effect of the membrane platform with biotin-streptavidin scaffold on PSA was evaluated by direct analysis of the mass tags using PSI-MS. The results show that on the membrane loaded with the scaffold (i.e.treatment of streptavidin and PSA antibody-modified biotin), the signal intensity of the mass label is more than 5 times higher than that of the bare membrane with only PSA antibody-treated biotin attached (FIGS. 3b, 3c and 3 d). It is demonstrated that the biotin-streptavidin treated nylon membrane (i.e., reaction platform) prepared in example 1 has excellent enrichment effect on PSA.

Example 8 comparison of the Effect of the Nylon Membrane blocking agent

The blocking of the reaction platform prior to detection of PSA is important to avoid non-specific interactions between the aptamer-containing probe and the membrane. The inventors compared the effect of several commonly used protein blocking agents (PBR) on membrane blocking, including BSA, fatty acid-free BSA and skim milk powder.

The lower the mass-labeled signal in the mass spectrometry results, indicating the better the membrane blocking before detection of PSA. Figure 4 shows that fatty acid-free BSA has the best blocking performance among the three PBRs.

Example 9 comparison and characterization of Probe preparation

The signal amplification of the probe designed by the application to the PSA depends on the load of the mass label on the gold nanoparticles. The inventors also compared the effect of different ratios of mass labels to aptamers on the signal output of AuNPs surfaces. It can be appreciated that the more mass tags that are combined with AuNP, the better the signal amplification. However, too much mass label attached to AuNPs may also prevent probes from binding to PSA^2cIn the absence of any antigen binding.

1mL of AuNPs (about 2 × 10) containing 0.1nmol of aptamer was used¹¹Particles) were optimized, the concentration of the mass labels varied from 0.125 to 25 nmol. FIG. 5 shows the mass spectrum signalThe highest mass label dosage of 20 nmol. In the subsequent analysis, 0.1nmol of aptamer, 20nmol of mass tag and 1mL of AuNPs were used to synthesize the probe.

The time at which TCEP activates the aptamer was also compared, which affects the binding of the aptamer to AuNP. TCEP can reduce the binding of the S-S bond of the aptamer to the-SH bond, thereby facilitating the binding of the aptamer to AuNPs. This application has tested the literature^2bThe 1h activation time reported in (1), unexpectedly, the AuNPs turned purple and aggregated. Thus, the inventors tried to shorten the activation time (1, 5, 15 and 30 minutes), and surprisingly found that agglomeration of aunps could be avoided even with shorter activation time treatments. Figure 6 shows that the activation time can be 1-30, significantly shorter than the 1 hour activation time reported in the literature. Wherein the signal of the quality label is strongest when the activation time is as short as 5 minutes. This may be related to AuNP (30nm) of a particular particle size and aptamer of a particular sequence as used herein.

The prepared probes were characterized using TEM to observe the appearance of Au and Au-Apt particles and further confirm Apt-modified aunps with UV-Vis. TEM images show that Apt-assembled Au-Apt particles are better dispersed in aqueous solution than bare AuNPs (fig. 7a and 7 b). The UV-Vis spectra show that conjugation between AuNP and aptamer results in a red shift of the absorption maximum from 524nm to 532nm (fig. 7 c). These results indicate that aptamers do bind to the surface of AuNPs. A red shift from 532nm to 536nm was also observed in the prepared Apt-AuNPs-TPTM probe compared to Apt-AuNPs, indicating that the mass label is indeed bound to the Au-Apt.

Example 10 comparison of the influencing factors of Mass Spectrometry

Efficient dissociation of the mass tag (TPTM) from the AuNP is also important to achieve good assay performance.

The present application contemplates the linkage between the major components of the reaction platform, such as the biotin-streptavidin-biotin scaffold, the immunoaffinity of the antibody to PSA, PSA and specific aptamers, and the Au-S bond of Apt-AuNP and TPTM-AuNP in the probe. These connections are kept stable until MS analysis is performed.For example, the dissociation constant of biotin from streptavidin is as low as femtomolar³Such stability is ensured from one side.

Thus, in order for the probe to release the mass label at the time of sample introduction, the previous connection of any two components described above may be broken. The inventors chose to release the mass label quantitatively by breaking the Au-S bond between the AuNP and the mass label. Mass labels cannot be ionized by high pressure alone due to the strong covalent interactions between the probe and PSA. The inventors tried to compare different substances added to the spray solvent to make the probe release easier.

The inventors compared the reducing agent (DDT) and the oxidizing agent (H)₂O₂) Acid (citric acid) 3 different treatments.

FIG. 8 shows the results of oxidation (H)₂O₂) The reduction treatment (DDT) was most efficient and the MS signal of TPTM was stronger than the acidification (citric acid) treatment. DDT in spray solvent dissociates the mass labels by a hydrogenation process.

In addition, in the signal mode of the quality label, two DDT dropping modes (direct dropping and pre-dropping) of the film were compared. Figure 10b shows that direct dropping can last several minutes of stable MS signal output. In contrast, the signal pre-dropped onto the membrane remained for only 10 seconds (fig. 10 c). Although the pre-drop mode may allow sufficient reaction time for the DTT and TPTM, it may also result in oxidation of the DTT, thereby reducing the dissociation efficiency of the mass label from the probe.

The inventors also compared ionized adduct products released by mass tag reduction in positive ionization mode of mass spectrometry.

The inventors have found that alkali metal salts (e.g., NH)₄OH) promotes the production of adducts containing mass tag monomers, thereby increasing sensitivity. FIG. 9 shows that both TPTM and IS can be reacted with NH₄ ⁺Cationic interaction to form [ M + NH ]₄]⁺Adduct, and [ M + NH₄]⁺The strength of the adduct is significantly higher than the corresponding [ M + Na ]]⁺And [ M + H]⁺An adduct. Further optimizing the NH added in the spray solvent₄OH concentration to achieve optimum ionization performance. FIG. 10a shows that the signal intensity of the mass label is at NH₄OH concentration was highest at 2M. The results show that addition of appropriate amount of NH to the spray solvent₄OH aids in the detection of mass labels, while excess NH₄OH decreases the signal intensity.

Example 11

1. Specificity and Cross-reactivity

The application also contrasts PSA analysis in authentic serum samples with other types of cancer-associated antigens to assess the specificity and cross-reactivity of the method.

Two serum glycoprotein antigens (CEA and CA125) commonly used in clinical assays were selected and PSA, CEA or CA125 antigens with the same concentration were added to the serum samples, either alone or in combination.

FIG. 11 shows that the signal intensity of PSA alone is comparable to that of mixed CEA or CA125, and that the signal intensity of CEA or CA125 alone is low. These results indicate that the reaction platform constructed herein has good selectivity and specificity for PSA even in the presence of other antigens.

2. Linear range, limit of detection, reproducibility

From 10pg mL^-1To 50ng mL^-1A good linear correlation between the intensity ratio and PSA concentration in PBST buffer (R) was observed over almost three orders of magnitude over a wide concentration range of (b) and (c) and (d) was observed²0.998) (fig. 12). LOD as low as about 4.0pg mL according to IUPAC recommendations, i.e., the standard deviation of a triple blank measurement^-1. In clinical diagnosis, if the concentration of PSA in serum is greater than 4ng mL^-1The patient may be at risk for prostate cancer. Thus, the methods of the present application are fully capable of meeting the clinical diagnostic needs of PSA measurements.

The reproducibility and stability of the established reaction platform on the membrane was evaluated with membranes having different shelf lives (1 to 30 days at 4 ℃). FIG. 13 shows that the analysis results for all membranes are consistent for at least 30 consecutive days with a relative standard deviation of 3.9% (1ng mL)^-1) And 1.8% (10ng mL)^-1)。

3. Recovery rate of added standard

Spiking recovery of different levels of PSA in human serum achieved spiking recoveries between 94% and 102%, with the results shown in table 3, indicating that the method of the present application is not affected by complex matrices of human serum.

TABLE 3 serum spiking recovery results for PSA

Comparative example 1

The method of the present application was compared in terms of analytical performance on PSA with other methods reported in the literature previously, as shown in table 4. The linear range and LOD of the method of the present application are comparable to most analytical methods and much better than MS-based methods. Although some electrode-based methods have good sensitivity, expensive electrodes limit the application of these methods to clinical analysis of disease biomarkers. In addition to low cost, the nylon membrane-based reaction platform of the examples of the present application has better durability and stability than cellulose paper commonly used in conventional PSI-MS.

TABLE 4 comparison of Performance of different PSA immunoassay methods

^aIndicating that the corresponding data are not mentioned in the literature.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the application and should not be taken as limiting the scope of the application. Rather, the scope of the application is defined by the appended claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Reference to the literature

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2.(a)Xu,S.T.；Ma,W.；Bai,Y.；Liu,H.W.,Ultrasensitive Ambient MassSpectrometry Immunoassays:Multiplexed Detection of Proteins in Serum and onCell Surfaces.Journal of the American Chemical Society 2019,141(1),72-75；(b)Du,R.J.；Zhu,L.N.；Gan,J.R.；Wang,Y.N.；Qiao,L.；Liu,B.H.,Ultrasensitive Detectionof Low-Abundance Protein Biomarkers by Mass Spectrometry Signal AmplificationAssay.Analytical Chemistry 2016,88(13),6767-6772；(c)Zhong,X.Q.；Qiao,L.；Gasilova,N.；Liu,B.H.；Girault,H.H.,Mass Barcode Signal Amplification forMultiplex Allergy Diagnosis by MALDI-MS.Analytical Chemistry 2016,88(12),6184-6189.

3.Haes,A.J.；Van Duyne,R.P.,A nanoscale optical blosensor:Sensitivityand selectivity of an approach based on the localized surface plasmonresonance spectroscopy of triangular silver nanoparticles.Journal of theAmerican Chemical Society 2002,124(35),10596-10604.

4.Garcia-Cortes,M.；Fernandez-Arguelles,M.T.；Costa-Fernandez,J.M.；Sanz-Medel,A.,Sensitive prostate specific antigen quantification usingdihydrolipoic acid surface-functionalized phosphorescent quantumdots.Analytica Chimica Acta 2017,987,118-126.

5.Gutierrez-Zuniga,G.G.；Hernandez-Lopez,J.L.,Sensitivity improvementof a sandwich-type ELISA immunosensor for the detection of differentprostate-specific antigen isoforms in human serum using electrochemicalimpedance spectroscopy and an ordered and hierarchically organizedinterfacial supramolecular architecture.Anal Chim Acta 2016,902,97-106.

6.Wei,Y.Y.；Wang,D.N.；Zhang,Y.Z.；Sui,J.H.；Xu,Z.R.,Multicolor andphotothermal dual-readout biosensor for visual detection of prostate specificantigen.Biosensors&Bioelectronics 2019,140,48-56.

7.Chong,J.；Chong,H.；Lee,J.H.,A chemiluminescent dual-aptasensorcapable of simultaneously quantifying prostate specific antigen and vascularendothelial growth factor.Anal Biochem 2019,564-565,102-107.

8.E.W.Klee,O.P.Bondar,M.K.Goodmanson,S.A.Trushin,R.J.Singh,N.L.Anderson,G.G.Klee,American Journal of Clinical Pathology 2014,141,527-533.

9.Lang,R.；Leinenbach,A.；Karl,J.；Swiatek-de Lange,M.；Kobold,U.；Vogeser,M.,An endoglycosidase-assisted LC-MS/MS-based strategy for theanalysis of site-specific core-fucosylation of low-concentrated glycoproteinsin human serum using prostate-specific antigen(PSA)as example.Clinica ChimicaActa 2018,480,1-8.

10.Chen,Y.T.；Tuan,L.P.；Chen,H.W.；Wei,I.A.；Chou,M.Y.；Chen,H.M.；Tyan,Y.C.；Chen,S.F.,Quantitative Analysis of Prostate Specific Antigen IsoformsUsing Immunoprecipitation and Stable Isotope Labeling MassSpectrometry.Analytical Chemistry 2015,87(1),545-553.

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Claims

1. A reaction platform comprising a support carrying directly or indirectly a capture element.

2. The reaction platform of claim 1, wherein an enrichment element is further carried between the support and the capture element;

preferably, the enrichment element is selected from at least one of biotin, streptavidin, avidin;

preferably, the enrichment element is selected from at least one of biotin, streptavidin;

preferably, the enrichment element is selected from biotin and streptavidin.

3. The reaction platform of claim 1, wherein the capture element is selected from at least one of an antibody or an aptamer;

preferably, the capture element comprises an antibody;

preferably, the support bears, or is modified to bear, at least one of the following groups: amino, carboxyl, aldehyde, epoxy; preferably, the support bears, or is modified to bear, an amino group;

preferably, the support is selected from at least one of nylon, electrostatic filament, cellulose paper, glass, chip, aluminum foil, stainless steel sheet;

preferably, the support is selected from nylon; preferably, the nylon is selected from Biodyne B nylon.

4. A probe comprising a metal nanoparticle having a mass tag and a capture element attached thereto.

5. The probe of claim 4, wherein the mass tag is selected from the group consisting of a compound having a thiol group;

preferably, the mass label is selected from at least one of trimethylolpropane tris (3-mercaptopropionate), N- (2-mercaptopropionyl) glycine, sodium 2-mercaptoethanesulfonate, sodium 3-mercapto-1-propanesulfonate, 4-mercaptophenylboronic acid, 3-mercapto-3-methyl-1-butanol, 4-mercaptophenol, 3-mercaptobenzoic acid, isooctyl 3-mercaptopropionate, butyl 3-mercaptopropionate, isooctyl thioglycolate, 6-mercaptopyridine-3-carboxylic acid;

preferably, the mass label is selected from trimethylolpropane tris (3-mercaptopropionate);

preferably, the capture element is selected from at least one of an antibody or an aptamer;

preferably, the capture element is selected from an aptamer;

preferably, the sequence of the aptamer is: 5' -SH- (CH)₂)₆-TTT ATT AAA GCT CGC CAT CAA ATA GCTTT-3'；

Preferably, the metal nanoparticles are selected from at least one of gold nanoparticles, silver nanoparticles, or platinum nanoparticles;

preferably, the metal nanoparticles are selected from gold nanoparticles;

preferably, the size of the metal nanoparticles is 20-50 nm; more preferably 30 nm;

preferably, the ratio of the number of the metal nanoparticles to the concentration of the mass label and the capture element is (1.8-2.5) × 10¹¹1 (1.25-250) and preferably in a ratio of 2 × 10¹¹：200:1。

6. A kit or reagent for protein detection comprising the reaction platform according to any one of claims 1 to 3 and/or the probe according to any one of claims 4 to 5.

7. A method for detecting a protein, comprising the steps of:

s1, placing the reaction platform of any one of claims 1 to 3 in a sample to be tested, and reacting for 1 to 2 hours at room temperature;

s2, after the step S1 is finished, mixing the probe with any one of claims 2 to 5, and continuing to react for 1 to 2 hours;

8. The detection method of claim 7, wherein the mass spectrometry is selected from the group consisting of open mass spectrometry;

preferably, the open mass spectrum is selected from paper spray open mass spectrum;

preferably, the spray solvent of the paper spray open mass spectrometry contains at least one of hydrogen peroxide, citric acid or DL-dithiothreitol;

preferably, the spray solvent contains hydrogen peroxide or DL-dithiothreitol;

preferably, the spray solvent contains DL-dithiothreitol;

preferably, the spray solvent is applied by direct dropwise addition;

preferably, the spray solvent further contains an alkali metal salt;

preferably, the alkali metal salt is selected from NH₄OH; preferably, NH₄The concentration of OH is 0.1-3M; more preferably 2M.

9. The detection method of claim 7, further comprising the steps of preparing a reaction platform and a probe;

preferably, the preparation of the reaction platform comprises the following steps:

3) incubating the support prepared in step 3) in a blocking agent;

preferably, the support is selected from a shape having at least one corner; preferably, the angle of the angle is selected from 20 ° -150 °;

preferably, the angle of the angle is selected from 50 ° -120 °; more preferably 60 °;

preferably, the blocking agent is selected from at least one of bovine serum albumin, fatty acid-free bovine serum albumin or skimmed milk powder;

preferably, the blocking agent is selected from fatty acid free bovine serum albumin;

preferably, the preparation of the probe comprises the following steps:

a) activating the capture element;

b) attaching a capture element to the metal nanoparticle;

c) attaching a mass tag to the metal nanoparticles prepared in step b);

preferably, the capture element is activated with an activator in step a);

preferably, the activator is selected from tris (2-carboxyethyl) hydrophosphate;

preferably, the activator activates the capture reagent for a period of time ranging from 1 to 30 minutes; preferably 5 minutes.

10. Use of the reaction platform of claims 1-3, or the probe of claims 1-5, or the reagent or kit of claim 6, or the detection method of claims 7-9 for detecting proteins;

preferably, the sample against which the detection is directed is selected from at least one of blood, urine, interstitial fluid, lymph fluid, cerebrospinal fluid, aqueous humor;

preferably, the blood comprises serum, plasma;

preferably, the protein is selected from at least one of amino acids, polypeptides, glycoproteins;

preferably, the protein is selected from at least one of prostate specific antigen, carcinoembryonic antigen, sugar chain antigen 125, alpha fetoprotein, recombinant human neuron specific enolase, sugar chain antigen 153, squamous cell carcinoma antigen, P53 protein, human epididymin 4;

preferably, the protein is selected from prostate specific antigens.