Colorimetric and photo-thermal sensor as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biochemical sensing, in particular to a colorimetric and photo-thermal sensor and a preparation method and application thereof. The sensor comprises a target detection object specific capture antibody, Y-DNA (Y-deoxyribonucleic acid), linker-DNA, hemin, dopamine and H2O2 (hydrogen peroxide), one end of the Y-DNA contains a target detection object aptamer sequence, and the other two ends of the Y-DNA are connected with the linker-DNA; the Y-DNA is composed of Y1, Y2 and Y3, and the linker-DNA comprises a G4 sequence; after a target detection object captured by the antibody is combined with an aptamer sequence at one end of Y-DNA, co-incubating with linker-DNA and hemin to form micro-hydrogel; the micro-hydrogel catalyzes dopamine into polydopamine under the condition of H2O2, and a solution obtained through co-incubation is the colorimetric and photo-thermal sensor. The colorimetric photo-thermal sensor realizes visual and high-sensitivity detection of the tumor marker PSA.

Description

Technical Field

[0001] The present invention relates to the field of biochemical sensing technology, and in particular to a colorimetric and photothermal sensor, a preparation method thereof, and an application thereof. Background Art

[0002] Malignant tumors are complex diseases that occur in all age groups, across multiple tissues and organs, and can lead to serious consequences. They have become one of the most serious threats to human health. With the rapid advancement of precision medicine, sensitive and quantitative analysis of tumor biomarkers has become increasingly important. Simultaneously, to address the challenge of highly sensitive biomarker analysis, scientists have developed a variety of sophisticated imaging and detection technologies over the past decade. These technologies typically utilize nanomaterials such as AuNPs and quantum dots in conjunction with advanced sensor devices to achieve highly sensitive detection of DNA or proteins. DNA nanotechnology, such as PCR, can also be combined to enhance detection sensitivity. Despite these remarkable achievements in biotechnology laboratories, widespread adoption of these technological innovations in biological and clinical laboratories remains limited due to the demand for specialized reagents and equipment. Colorimetric sensors, a sensory signal transduction platform capable of visual measurement, have been a crucial diagnostic tool for nearly a century. However, traditional colorimetric diagnostic methods are limited in sensitivity and specificity by the influence of human visual perception. Therefore, the development of simple and sensitive colorimetric sensors is particularly urgent for point-of-care testing and clinical diagnosis.

[0003] Enzyme-linked immunosorbent assays (ELISAs) are currently widely used to detect trace biomarkers. Their primary detection principle is to organically combine the antigen-antibody immune reaction with the efficient catalytic action of enzymes. The enhanced amplification effect of enzyme-catalyzed substrates reveals the nature and content of the antigen or antibody in the reaction system. Horseradish peroxidase (HRP), the most commonly used enzyme in in vitro diagnostic reagents, suffers from drawbacks such as difficulty in labeling, easy inactivation, and instability. G4 / hemin nuclease, composed of hemin chloride and a G-quadruplex structure, is a promising candidate for HRP mimics. Compared to native HRP, HRP-mimicking DNA enzymes are easier to prepare, more cost-effective, and more stable. G4 / hemin has been widely used in colorimetric, chemiluminescent, and electrochemical assays, and therefore holds broad application prospects in the biomedical field. DA (dopamine), a commonly used coating material in surface treatment, has also been found to serve as a catalytic substrate for HRP. Research has shown that PDA (polydopamine) can be site-specifically deposited near HRP, achieving more efficient colorimetric results. Inspired by the fact that polyaniline formed by G4 / hemin-catalyzed aniline deposition on DNA chains, we wondered whether polydopamine formed by G4 / hemin-catalyzed dopamine deposition on DNA chains could achieve a more efficient colorimetric effect. Currently, no technical reports have been found. DNA nanohydrogels are an emerging nanomaterial formed by the self-assembly of DNA molecules. Our research found that DNA nanohydrogels containing G4 / hemin structures can also increase the specific deposition of PDA and exhibit a more efficient colorimetric response. In addition, PDA can effectively absorb light and convert it into heat, which can be transferred to the surrounding environment and cause the temperature to rise. The temperature signal caused by the photothermal effect can be easily detected by a thermometer with high sensitivity without the need for advanced analytical instruments.

[0004] Based on this, this study developed a colorimetric and photothermal sensor. By constructing micro-hydrogels, G4 / hemin on the micro-hydrogels catalyzed DA polymerization to produce color and temperature changes, thereby realizing the detection of biomarkers, which is expected to produce more reliable and accurate results. Summary of the Invention

[0005] The present invention provides a colorimetric and photothermal sensor and a preparation method and application thereof, which realizes visual and highly sensitive detection of the tumor marker PSA and solves the problems existing in the prior art.

[0006] One of the technical solutions adopted by the present invention is: Provided is a colorimetric, photothermal sensor comprising a target detectable substance-specific capture antibody, Y-DNA, linker-DNA, hemin, dopamine, and H2O2; One end of the Y-DNA contains the target detection object adapter sequence, and the other two ends of the Y-DNA are connected to the linker-DNA respectively; the Y-DNA is composed of three DNA chains Y1, Y2 and Y3, and the linker-DNA is a linker DNA containing the G4 sequence; The target detected object captured by the target detection object-specific capture antibody binds to the target detection object aptamer sequence at one end of the Y-DNA, and is then co-incubated with the linker-DNA and hemin to self-assemble into a micro-hydrogel; the micro-hydrogel catalyzes dopamine to form polydopamine and deposits polydopamine, and the solution obtained by adding H2O2 and co-incubating is the colorimetric and photothermal sensor; Among them, the DNA sequence of Y1 is shown as SEQ ID NO.1, the DNA sequence of Y2 is shown as SEQ ID NO.2, and the DNA sequence of Y3 is shown as SEQ ID NO.3; the sequence of linker-DNA is shown as SEQ ID NO.4.

[0007] Furthermore, the sequences of the above Y-DNA and linker-DNA are as follows: Y1 SEQ ID NO.1: 5′-AAT TAA AGC TCG CCA TCA AAT AGC TTT AAG ATG ATT CTG TAG ATC TGT CGA TGA A -3′ (the underlined part is the PSA aptamer sequence).

[0008] Y2 SEQ ID NO.2: 5'-CCACAC CCG ATC TTC ATC GAC AGA TCC AGT CGC TGT ACTG-3'.

[0009] Y3 SEQ ID NO.3: 5′-CCACAC CCG ATC CAT GAC AGC GAC TGT ACA GAA TCA TCTT-3′.

[0010] linerDNA SEQ ID NO.4: 5′-GATCGG GTG TGG TTT GGG AAA GGG AAA GGGAAA GGG TTT GGT GTG GGC TAG-3′ (the underlined part is the G4 sequence).

[0011] Furthermore, the above colorimetric and photothermal sensors are solutions for performing colorimetric and photothermal analysis.

[0012] Furthermore, the target detection object is PSA or a biological sample containing PSA.

[0013] Furthermore, the biological sample containing PSA is a blood sample containing PSA.

[0014] Furthermore, the colorimetric and photothermal sensor further comprises a blocking buffer, a PBS buffer and a PBST washing solution.

[0015] Furthermore, the blocking buffer is PBS buffer supplemented with 1% BSA.

[0016] Furthermore, the PBST washing solution is incubated overnight in a 96-well plate with a specific capture antibody for the target detection object, and then the plate is washed; after washing, it is blocked with a blocking buffer; then the plate is washed with PBS, the target detection object is added, and the wells are washed again with PBST washing solution after continuing to incubate; then the Y-DNA, linker-DNA and hemin are added and incubated, and after incubation, the plate is washed and H2O2 is added and incubated again to obtain a solution for colorimetric and photothermal analysis.

[0017] Furthermore, an aqueous solution control is set up in the above treatment process for comparison with the colorimetric and photothermal sensors.

[0018] Furthermore, the colorimetric and photothermal sensors perform absorbance spectrum measurement within 450-700 nm to observe color changes; when the colorimetric and photothermal sensors are exposed to 808 nm near-infrared laser light at 1 W / cm 2 After vertical irradiation with a power density of 10 minutes, the temperature change was recorded.

[0019] Furthermore, the above temperature changes were recorded using a thermocouple microprobe.

[0020] Furthermore, when the target substance is present, the solution turns brown within 3 minutes; ... 2 After 10 minutes of vertical irradiation with a power density of 100 nm, the temperature increased significantly.

[0021] The second technical solution adopted by the present invention is: A method for preparing the above-mentioned colorimetric and photothermal sensors is provided, comprising the following steps: S1. Incubate the carboxyl-modified 96-well plate with a certain concentration of target-specific capture antibody at 4°C overnight. S2. Rinse the plate with PBST and block with 1% BSA in PBS at 37°C for 1 hour. S3. Wash the plate three times with PBS buffer, then add the target assay and incubate at 37°C for 1 hour; wash the wells again three times with PBST; S4, introduce Y-DNA, linker DNA and hemin, and incubate at 37°C for another 1 hour; S5. Wash the plate three times, add DA (dopamine) and H2O2, and incubate at 37°C for 3 minutes; the resulting solution is used for colorimetric and photothermal analysis.

[0022] Furthermore, the target detection object is PSA or a biological sample containing PSA; the target detection object specific capture antibody in S1 has a concentration of 20 μg·mL -1 PSA-specific capture antibody; the amount of target detection substance in S3 is 100 μL; Y-DNA, linker DNA and hemin with a total volume of 100 μL are introduced into S4, and the final concentrations of Y-DNA, linker DNA and hemin are all 5 μM; the amount of DA and H2O2 added in S5 is 100 μL (equal volume ratio), and the final concentration of H2O2 is 25 mM-75 mM. Furthermore, the above-mentioned H2O2 concentration is preferably 50mM.

[0023] Furthermore, the DA concentration was 10 mg·mL -1 , 15 mg·mL -1 , 20 mg·mL -1 ; preferably 15 mg·mL -1 .

[0024] Furthermore, the reaction pH in the above S5 is 8.5.

[0025] The third technical solution adopted by the present invention is: Provided is the application of the above-mentioned colorimetric and photothermal sensors in the visual sensitive detection of PSA in samples for non-diagnostic purposes.

[0026] Furthermore, the sample is a human serum sample.

[0027] Furthermore, the detection limit of PSA in the sample by the colorimetric and photothermal sensors was 0.51 ng·mL -1 .

[0028] Beneficial effects of the present invention: 1. The colorimetric and photothermal sensor of the present invention achieves visual and highly sensitive detection of the tumor marker PSA through ultrafast deposition of polydopamine.

[0029] 2. The present invention constructs a Y-DNA containing a PSA aptamer sequence at one end, and the other two ends can be connected to another Y-DNA through a linker-DNA containing a G4 sequence. In this way, after the incorporation of hemin, a microhydrogel is formed. The microhydrogel containing G4 / hemin catalyzes dopamine, causing ultra-fast deposition of polydopamine, ultimately achieving a more efficient colorimetric response, generating color and temperature changes to realize reliable and accurate detection of the biomarker PSA. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the ultrafast deposition of polydopamine on the surface of DNA micro-hydrogel and the photothermal detection of PSA; Figure 2 Characterization of DNA microhydrogel formation and G4 / hemin-catalyzed DA oxidative aggregation; Figure 3 This is the AFM imaging of pure L-DNA catalyzing dopamine to form polydopamine; Figure 4 Colorimetric and photothermal immunoassays for PSA of the present invention; Figure 5 For dual readout immunoassays by color and temperature; Figure 6 Selectivity for immunoassay detection of PSA; Figure 7 Colorimetric and photothermal immunoassays for serum PSA; Figure 8 To investigate the effect of H2O2 concentration on the detection effect in the preparation method of the present invention; Figure 9 To investigate the effect of DA concentration on the detection effect in the preparation method of the present invention; Figure 10 To investigate the effect of solution reaction pH on the detection effect in the preparation method of the present invention; Figure 11 To investigate the effect of anti-PSA concentration on the detection effect in the preparation method of the present invention; Figure 12 This study investigates the effect of Y-DNA concentration on the detection effect in the preparation method of the present invention.

[0031] in, Figure 2 Figure A is a natural PAGE demonstration of the synthesis of DNA nanohydrogels; Figure B is an AFM image of DNA microhydrogels catalyzing the conversion of DA to PDA; Figure C is a UV-visible spectrum of G4 / hemin-catalyzed DA oxidation to produce PDA; Figure D is a temperature curve of G4 / hemin-mediated DA oxidation to form PDA. Figure 4A is the UV-vis curve corresponding to the reaction of DA on different samples by incubation with anti-PSA (a), anti-PSA + PSA (b), anti-PSA + BSA + Y-DNA (c), anti-PSA + Y-DNA + L-DNA (d), and anti-PSA + PSA + Y-DNA + L-DNA (e); B is the temperature curve of DA on different samples by incubation with anti-PSA (a), anti-PSA + PSA (b), anti-PSA + BSA + Y-DNA (c), anti-PSA + Y-DNA + L-DNA (d), and anti-PSA + PSA + Y-DNA + L-DNA (e); Figure 5 A is the UV-vis curve of DA solution at different PSA concentrations (from a to h); B is the UV-vis absorption intensity as a function of PSA concentration, and the lower right inset is the UV-vis absorption intensity as a function of PSA concentration; C is the temperature curve of DA dispersion at different PSA concentrations (1, 5, 10, 50, 100, 200, 500 and 1000 ng·mL-1) under 808 nm laser irradiation (from a to h); D is the function of temperature and PSA concentration, and the lower right inset is the linear relationship between temperature and the logarithm of PSA concentration; Figure 6 a corresponds to 100 ng·mL -1 CEA, b corresponds to 100 ng·mL -1 AFP, c corresponds to 100 ng·mL - 1 thrombin, d corresponds to 100 ng·mL -1 APE1, e corresponds to 100 ng·mL -1 PSA, f corresponds to 100 ng·mL -1 PSA samplecoexisted with 100 ng·mL -1 CEA, 100 ng mL -1 AFP, 100 ng mL -1 thrombin, and 100 ng·mL -1 APE1; Figure 7A in the middle shows the UV-vis absorption spectra of DA solutions measured in the presence of different concentrations of PSA; the upper inset shows the immunoassay of PSA in serum samples through color readout; B shows the UV-vis absorption intensity as a function of PSA concentration, and the inset shows the linear relationship between the peak absorption intensity and the logarithm of the PSA concentration; C analyzes the serum temperature curve of DA dispersions with different PSA concentrations under 808 nm irradiation; D shows the function of temperature and PSA concentration, and the inset shows the linear relationship between temperature and the logarithm of PSA concentration; PSA concentrations (ah) are: 1, 10, 50, 100, 200, 500, 750, and 1250 ng·mL -1 ; Figures 8-12 From left to right in the figure, it corresponds to the order of adjusting variables in the corresponding embodiment. DETAILED DESCRIPTION

[0032] In order to clearly illustrate the technical features of this solution, the present invention is described in detail below through specific implementation methods in conjunction with the accompanying drawings.

[0033] In the present invention, the equipment, raw materials and reagents used can be purchased from the market or are commonly used in the art. The detection methods in the following examples are all conventional methods in the art unless otherwise specified.

[0034] Materials, reagents and sources used in this example experiment: All oligonucleotides were obtained from Bioengineering Technology Services Co., Ltd. All synthetic oligonucleotides were HPLC-purified and freeze-dried by the supplier. Dopamine and hydrogen peroxide (H₂O₂) were purchased from Sigma-Aldrich. Anti-PSA antibody (ab182031) was purchased from Abcam. Total prostate-specific antigen (PSA), carcinoembryonic antigen (CEA), fetoprotein (AFP), thrombin, and apurinic / apyrimidinic endonuclease 1 (APE1) were purchased from Biocell Company (Zhengzhou, China). All other chemical reagents were of analytical grade. Carboxyl-modified 96-well plates were purchased from Corning. Phosphate buffer (1× PBS) consists of 10 mM phosphate-buffered saline, 50 mM NaCl, and 30 mM KCl (pH 8.5). Blocking buffer consisted of 1× PBS supplemented with 1% (w / v) bovine serum albumin (pH 8.5). The washing solution (PBST) consisted of 1× PBS solution (pH 8.5) and 0.05% (v / v) Tween 20. All solutions were prepared using ultrapure water (Millipore).

[0035] Example 1 PSA immunoassay A method for preparing a colorimetric and photothermal sensor for visualizing and highly sensitive detection of the tumor marker PSA comprises the following steps: S1. Place the carboxyl-modified 96-well plate in a 20 μg mL -1 The cells were incubated with PSA-specific capture antibody (anti-PSA antibody ab182031) at 4°C overnight. S2. Rinse the plate with PBST and block with 1% BSA in PBS at 37°C for 1 hour. S3. Wash the plate three times with PBS buffer, then add 100 μL of target assay and incubate at 37°C for 1 hour. Wash the wells again three times with PBST. S4. Introduce a total of 100 μL of Y-DNA, linker DNA, and hemin to a final concentration of 5 μM each; incubate at 37°C for another 1 hour to form a DNA microhydrogel; S5. Wash the plate three times and add 100 μL of DA and H2O2 (volume ratio 1:1), where the final concentration of DA is 15 mg mL -1 , the final H2O2 concentration is 50mM; the pH is controlled at 8.5, and incubated at 37°C for 3 minutes; the resulting solution is a colorimetric and photothermal sensing system, which can be used for colorimetric and photothermal analysis to achieve efficient detection of PSA.

[0036] The above colorimetric analysis is to measure the absorbance spectrum in the range of 450nm to 700nm. The photothermal measurement is to irradiate the solution with 808nm near infrared (NIR) laser at 1W / cm 2 The power density was irradiated vertically for 10 minutes. The temperature change was recorded by a thermocouple microprobe, and water was used as a control solution.

[0037] In the above preparation steps, the sequence of the Y-DNA used is as follows: Y1 SEQ ID NO.1: 5′-AAT TAA AGC TCG CCA TCA AAT AGC TTT AAG ATG ATT CTG TAG ATC TGT CGA TGA A (PSA aptamer)-3′.

[0038] Y2 SEQ ID NO.2: 5'-CCACAC CCG ATC TTC ATC GAC AGA TCC AGT CGC TGT ACTG-3'.

[0039] Y3 SEQ ID NO.3: 5′-CCACAC CCG ATC CAT GAC AGC GAC TGT ACA GAA TCA TCTT-3′.

[0040] In the above preparation steps, the sequence of the linker DNA used is as follows: linerDNA SEQ ID NO.4: 5′-GAT CGG GTG TGG TTT GGG AAA GGG AAA GGGAAA GGG (G4 sequence) TTT GGT GTG GGC TAG-3′.

[0041] In the above preparation steps, the target detection substance is PSA or serum sample. The target detection substance PSA is added to realize the preparation of PSA immunosensor and complete PSA immunoassay; serum sample is added to realize PSA detection of clinical samples.

[0042] Example 2 The preparation method was the same as that in Example 1, except that the concentration of H2O2 in S5 was adjusted to 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, and 75 mM, respectively, to investigate the effect of H2O2 concentration on the detection effect.

[0043] The obtained colorimetric sensing system solution was subjected to colorimetric determination, see Figure 8 It can be seen that when the H2O2 concentration is 25 mM, 50 mM, and 75 mM, the color reaction is obvious, and 50 mM is preferred.

[0044] Example 3 The preparation method was the same as that in Example 1, except that the final concentration of DA was adjusted to 2.5 mg·mL -1 , 5 mg·mL -1 , 7.5 mg·mL -1 , 10 mg·mL -1 , 15 mg·mL -1 , 20 mg·mL -1 , investigate the effect of DA concentration on the detection effect.

[0045] The obtained colorimetric sensing system solution was subjected to colorimetric determination, see Figure 9 It can be seen that when the above DA concentration is 15 mg·mL -1 When the color reaction is obvious.

[0046] Example 4 The preparation method was the same as that in Example 1, except that the pH of the solution in step S5 was adjusted to 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, respectively, to investigate the effect of the solution pH on the detection effect.

[0047] The obtained colorimetric sensing system solution was subjected to colorimetric determination, see Figure 10 It can be seen that when the pH is 8.5, the color reaction is obvious.

[0048] Example 5 The preparation method was the same as that in Example 1, except that the concentration of anti-PSA was adjusted to 1 μg·mL -1 , 2.5 μg·mL -1 , 5 μg·mL -1 , 7.5 μg·mL -1 , 10 μg·mL -1 , 15 μg·mL -1 , 20 μg·mL -1 , 25 μg·mL -1 , to investigate the effect of anti-PSA concentration on the detection effect.

[0049] The obtained colorimetric sensing system solution was subjected to colorimetric determination, see Figure 11 It can be seen that when the anti-PSA concentration is 20 μg·mL -1 When the color reaction is obvious.

[0050] Example 6 The preparation method was the same as that in Example 1, except that the Y-DNA concentration was adjusted to 0.1 µM, 0.5 µM, 1 µM, 1.5 µM, 2.5 µM, 5 µM, 7.5 µM, and 10 µM, respectively. The effect of Y-DNA concentration on the detection effect was investigated.

[0051] The obtained colorimetric sensing system solution was subjected to colorimetric determination, see Figure 12 It can be seen that when the Y-DNA concentration is 5 μM, the color reaction is obvious.

[0052] Taking the colorimetric photothermal sensor prepared by the preparation method of Example 1 as an example, the following experimental verification was carried out.

[0053] 1. Construction of Microhydrogels (DNA Nanohydrogels) and Verification of Efficient Catalytic PDA Colorimetry and Photothermal Response 1.1 Verification of micro-hydrogel formation by gel electrophoresis The microhydrogel (DNA nanohydrogel) is formed by Y-DNA connected together by linker DNA. Y-DNA is composed of three DNA chains: DNA1, DNA2, and DNA3.

[0054] The interaction between Y-DNA and linker DNA was verified by native polyacrylamide gel electrophoresis (PAGE). As described in step S4 of the previous preparation, the reaction was performed by incubating Y-DNA with linker DNA (G4) at 37°C for 1 hour. After the reaction, the mixture was loaded onto an 8% native polyacrylamide gel electrophoresis (PAGE) and electrophoresed in 1× tris-borate-EDTA (TBE) buffer (containing 89 mM tris(hydroxymethyl)aminomethane, 2 mM ethylenediaminetetraacetic acid, and 89 mM boric acid, pH 8.0) at a constant voltage of 100 V for 2.5 hours. The gel was then stained with SYBR Green II for 30 minutes and visualized using the ChemiDoc™ MP system (Bio-Rad).

[0055] At the same time, replace "Y-DNA and linkerDNA" in the previous steps with DNA1, DNA2, DNA3, linkerDNA, DNA1+DNA2, DNA2+3, DNA1+3, and DNA1+2+3, respectively. Incubate the reactions at 37°C for 1 h. After the reaction, load them onto 8% native PAGE and perform visualization as above.

[0056] The above DNA groups were also prepared at the same final concentration as that in S4.

[0057] See also Figure 2 Figure A, native PAGE demonstrates the synthesis of DNA nanohydrogels. Band a is DNA1, band b is DNA2, band c is DNA3, and band d is linker DNA. It can be seen that the four DNA chains are all displayed as single bands in the gel image, indicating that none of the four DNA chains have complex secondary structures. Band e is the binding product of DNA1 and DNA2, band f is the binding product of DNA1 and DNA3, and band g is the binding product of DNA2 and DNA3. These three bands are significantly elevated in the gel image, but the bands have tailing phenomena, indicating that although DNA1, DNA2, and DNA3 can assemble in pairs, the binding efficiency is not high. Band h is the combination of DNA1+DNA2+DNA3. It can be seen that the DNA bands are significantly elevated, and the three DNA chains assemble to form Y-DNA, and the bands have no tailing phenomenon, indicating that the formed Y-DNA is uniform and stable. After adding linker DNA, as in band i, the DNA bands accumulate at the top of the gel, indicating the formation of microhydrogels.

[0058] 1.2 Verification of micro-hydrogel formation using AFM See also Figure 2 Middle B shows an AFM image of the micro-hydrogel efficiently catalyzing the formation of PDA from DA. Clear DNA assemblies are formed, and compared to single-stranded DNA, the assembly height decreases from 0.6 nm to 1.5 nm.

[0059] like Figure 3 , which is an AFM image of pure L-DNA catalyzing the formation of polydopamine (PDA) from dopamine (DA). It can be seen that the DNA micro-hydrogel formed in this example can achieve more efficient PDA deposition.

[0060] 1.3 UV-Vis spectroscopy of microhydrogels to verify the ability of linker DNA / hemin to catalyze DA See also Figure 2 The solutions in the three centrifuge tubes in the upper right corner of C, pure DA ( Figure 2 a) in C is a homogeneous and transparent solution. When linker DNA / hemin is added ( Figure 2 (b) in C), the solution turned brown after 3 minutes, indicating that DA rapidly polymerized. The polymerization was catalyzed by G4 / hemin to produce PDA. After adding Y-DNA again ( Figure 2 The color change of the solution is more obvious, indicating that the DNA nanohydrogel promotes the deposition of PDA. The UV absorption of the linker DNA / hemin catalyzed DA to form PDA is measured, as shown in Figure 3. Figure 2 From the three lower curves in Figure C, we can see that DA has no obvious UV absorption at 490 nm. When catalyzed by linker DNA, the UV absorbance at 490 nm is significantly enhanced, and after the addition of Y-shaped DNA, the UV absorbance increases again.

[0061] 1.4 Verification of the photothermal performance of G4 / hemin catalyzing DA to PDA See also Figure 2 In Figure D, pure DA has basically no temperature change under 808 nm laser radiation, as shown in curve a in the figure; after using linker DNA to catalyze the formation of PDA, the temperature increases significantly, as shown in curve b in the figure; when Y-DNA is added, the temperature increases again, as shown in curve c in the figure.

[0062] 2. Capability Verification of the Constructed Immunosensor for Detecting PSA Based on the preparation method of Example 1, anti-PSA, anti-PSA + PSA, anti-PSA + PSA + Y-DNA, anti-PSA + Y-DNA + linker DNA, and anti-PSA + PSA + Y-DNA + linker DNA were added to the surface of a carboxylic acid-modified 96-well plate in groups to prepare immunosensors, and the color changes were observed.

[0063] like Figure 4Figures A in the middle and a-e in the upper panels correspond to the color development of a 96-well plate after adding anti-PSA, anti-PSA + PSA, anti-PSA + PSA + Y-DNA, anti-PSA + Y-DNA + linker DNA, and anti-PSA + PSA + Y-DNA + linker DNA. As can be seen, the first three samples produced no color, indicating that anti-PSA, PSA, and Y-DNA do not catalyze PDA color development. Furthermore, in the absence of PSA, Y-DNA and linker DNA do not bind to the plate surface, nor does the solution exhibit a noticeable color change. Only after the addition of PSA does the solution color change significantly. UV absorbance also confirmed that G4 / hemin catalyzed DA to form poly-PDA. Figure 4 From the lower middle curve, we can see that only when anti-PSA+PSA+Y-DNA+linkerDNA (e) are present at the same time, the UV absorbance of the solution at 490 nm will change significantly. Figure 4 In Figure B, it can be seen that after 10 minutes of irradiation with 808 nm near-infrared light, there was no obvious temperature change in the anti-PSA, anti-PSA+PSA, anti-PSA+PSA+Y-DNA, and anti-PSA+Y-DNA+linkerDNA samples. Only the anti-PSA+PSA+Y-DNA+linkerDNA (e) had obvious photothermal changes, which is consistent with the results of the above colorimetric method.

[0064] The above results show that the immunosensor constructed in Example 1 can detect PSA in both colorimetric and photothermal modes.

[0065] 3. Sensitivity of immunosensor for detecting PSA Based on the preparation method of Example 1, different concentrations of PSA (target detection substance) were introduced: 1, 5, 10, 50, 100, 200, 500 and 1000 ng·mL -1 , record the solution color respectively.

[0066] See also Figure 5 , as the PSA concentration increases, the solution color gradually increases. Figure 5 Figure A shows the change in UV absorbance after adding the target PSA. It can be clearly seen that the UV absorbance change is 1 ng·mL -1 to 1000 ng·mL -1 The dynamic range of the assay showed a good linear relationship with the logarithmic value of PSA concentration, and the detection limit of PSA was calculated to be 0.63 ng·mL -1At the same time, the photothermal curves corresponding to different concentrations of PSA were studied, such as Figure 5 In Figure C, we can also see that as the PSA concentration increases, the solution temperature changes more significantly, and the temperature change has a good linear relationship with the logarithm of the PSA concentration, as shown in Figure 4. Figure 5 The detection limit of PSA was calculated to be 0.51 ng·mL. -1 To the best of our knowledge, the sensitivity of this colorimetric and photothermal sensor is better than that of most previously reported PSA sensors.

[0067] 4. Specificity of Immunosensors Other protein biomarkers with higher concentrations were used as interfering substances, such as CEA, AFP, thrombin, and APE1. Figure 6 As shown, based on the steps of Example 1, comparative experiments were performed using CEA, AFP, thrombin, and APE1 instead of PSA. It can be seen that only PSA can cause obvious color changes, while CEA, AFP, thrombin, and APE1 do not cause obvious color changes.

[0068] like Figure 6 Figure f also verifies the immunosensor's ability to detect PSA in an interfering environment. When 10 nM of CEA, AFP, and 10 nM of PSA were added to the solution, the solution turned brown, with no significant difference in color compared to the solution containing only PSA.

[0069] The above results show that the immunosensor constructed in Example 1 can achieve highly sensitive and specific PSA detection. The above experiments show that the proposed immunosensing strategy can provide high selectivity for biomolecule detection, making this immunosensing strategy have great potential in the accurate early diagnosis of cancer.

[0070] 5. ELISA for real clinical samples The proposed immunosensor was initially applied to real human serum samples to evaluate the analytical reliability and potential of the proposed method. Serum specimens from PSA-positive or -negative patients were collected from Linyi People's Hospital. The samples were centrifuged, aliquoted, and stored at -20°C until analysis. The detection protocol for clinical samples strictly followed the standard sandwich assay method, except that actual clinical serum samples from different patients were used in place of PSA in the previously described assay. All serum samples were diluted 1:20 before application to the assay plate.

[0071] like Figure 7 As shown in Figure 2, as the PSA concentration increases, the UV absorbance also gradually increases. At 1250 ng·mL -1 .Concentration reached a plateau. At 1ng·mL-1 to 1250 ng·mL -1 Within a wide concentration range, UV absorbance showed a linear relationship with the increase in PSA. Furthermore, with laser irradiation, the solution temperature gradually increased over time, showing a linear relationship with the increase in PSA concentration. This method demonstrated good recovery rates for authentic sample analysis, demonstrating the suitability of colorimetric and photothermal immunoassays for analyzing complex biological samples.

[0072] The above describes in detail the colorimetric and photothermal sensors, their preparation methods, and their applications provided by the present invention. The above specific embodiments are not intended to limit the scope of protection of the present invention. Those skilled in the art will appreciate that any modifications or variations to the embodiments of the present invention fall within the scope of protection of the present invention.

[0073] Any matters not described in detail in the present invention are well-known technologies to those skilled in the art.

Claims

1. A colorimetric and photothermal sensor, characterized in that: Includes target-specific capture antibodies, Y-DNA, linker-DNA, hemin, dopamine, and H2O2; One end of the Y-DNA contains the target detection object adapter sequence, and the other two ends of the Y-DNA are connected to the linker-DNA respectively; the Y-DNA is composed of three DNA chains Y1, Y2 and Y3, and the linker-DNA is a linker DNA containing the G4 sequence; The target detected object captured by the target detection object-specific capture antibody binds to the target detection object aptamer sequence at one end of the Y-DNA, and is then co-incubated with the linker-DNA and hemin to self-assemble into a micro-hydrogel; the micro-hydrogel catalyzes dopamine to form polydopamine under H2O2 conditions and deposits polydopamine, and the solution obtained by co-incubation is the colorimetric and photothermal sensor; Among them, the DNA sequence of Y1 is shown as SEQ ID NO.1, the DNA sequence of Y2 is shown as SEQ ID NO.2, and the DNA sequence of Y3 is shown as SEQ ID NO.3; the sequence of linker-DNA is shown as SEQ ID NO.

4.

2. The colorimetric and photothermal sensor according to claim 1, characterized in that: The target detection object is PSA or a biological sample containing PSA.

3. The colorimetric and photothermal sensor according to claim 1, characterized in that: Also included are blocking buffer, PBS buffer, and PBST wash buffer.

4. The colorimetric and photothermal sensor according to claim 1, characterized in that: The colorimetric and photothermal sensors measure absorbance spectra within 450-700 nm to observe color changes; when irradiated by an 808 nm near-infrared laser at 1 W / cm 2 After vertical irradiation with a power density of 10 minutes, the temperature change was recorded.

5. The colorimetric and photothermal sensor according to claim 4, characterized in that: When the target substance is present, the solution turns brown within 3 minutes; the temperature rises significantly after near-infrared laser irradiation.

6. The method for preparing the colorimetric or photothermal sensor according to any one of claims 1 to 5, characterized in that: The steps are as follows: S1. Incubate the carboxyl-modified 96-well plate with a certain concentration of target-specific capture antibody at 4°C overnight. S2. Rinse the plate with PBST and block with blocking buffer consisting of 1% BSA in PBS at 37°C. S3. Wash the plate three times with PBS buffer, then add the target assay and incubate at 37°C for 1 hour; wash the wells again three times with PBST; S4, introduce Y-DNA, linker DNA and hemin, and incubate at 37°C for another 1 hour; S5. Wash the plate three times, add dopamine and H2O2, and incubate at 37°C for 3 minutes; the resulting solution is used for colorimetric and photothermal analysis.

7. The preparation method according to claim 6, characterized in that The target detection object is PSA or a biological sample containing PSA; the target detection object specific capture antibody in S1 has a concentration of 20 μg·mL -1 PSA-specific capture antibody; the amount of target detection substance in S3 is 100 μL; Y-DNA, linker DNA and hemin with a total volume of 100 μL are introduced into S4; the amount of dopamine and H2O2 added in S5 is 100 μL, and the final concentration of H2O2 is 25 mM-75 mM.

8. The preparation method according to claim 7, characterized in that The H2O2 concentration was 50 mM; the dopamine concentration was 15 mg·mL -1 .

9. Use of the colorimetric or photothermal sensor according to any one of claims 1 to 5 for visually sensitive detection of PSA in samples for non-diagnostic purposes.

10. The use according to claim 9, characterized in that The sample is a human serum sample; the detection limit of PSA in the sample detected by the colorimetric and photothermal sensors is 0.51 ng·mL -1 .

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