Candida albicans specific detection kit and application thereof

Abstract

This invention discloses a specific detection kit for Candida albicans and its application. The kit includes a nanoprobe, which is based on gold nanostars, with Raman probe molecules and Candida albicans-specific aptamers modified on the substrate surface. The kit exhibits high sensitivity, specificity, reproducibility, uniformity, and stability. Raman spectra can be acquired using a portable Raman spectroscopy device, and it can be used for the analysis of trace amounts of Candida albicans in samples, with a detection limit of 1.14–1.21 CFU / mL. Compared with traditional methods, the detection method of this kit has advantages such as simplified pretreatment procedures, shorter detection time, and simpler operation, significantly reducing time and economic costs, and is more suitable for rapid on-site detection of Candida albicans.

Description

Technical Field

[0001] This invention belongs to the field of biomedical analysis and detection, and in particular relates to a specific detection kit for Candida albicans and its application. Background Technology

[0002] Candida albicans ( Candida albicans Candida albicans is a common food spoilage microorganism, particularly suited to growing and multiplying in high-sugar, acidic environments. It is commonly found in yogurt, canned fruit, fruit juice, and tomato sauce. Its contamination not only leads to product rancidity and spoilage but also produces unpleasant odors, causes packaging bulging (due to gas production), and damages the original texture and taste of the food. This type of microbial activity severely impacts product quality, resulting in significant economic losses and food resource waste. Candida albicans causes incurable mastitis in dairy cows, causing not only a sharp drop in milk production and suffering but also an abnormally high somatic cell count in the milk, severely degrading its quality. If this contaminated raw milk enters the processing stage, it will directly contaminate dairy products such as milk, yogurt, and cheese. Its secreted metabolites and the resulting microbial contamination can accelerate food spoilage, threatening consumer health, especially posing a potential risk to immunocompromised individuals, ultimately causing huge economic losses to the entire dairy industry chain from breeding to production.

[0003] Existing methods for detecting Candida albicans have certain limitations. For example, microscopic examination, although rapid, has extremely low sensitivity, only effective when the bacterial load in clinical samples is sufficiently high (>10). 4 Candida albicans can only be observed at concentrations of CFU / mL, and it cannot distinguish it from other Candida species (such as Candida glabrata and Candida tropicalis). It is highly dependent on the subjective experience of the testing personnel, making it prone to missed detections and misdiagnosis. Immunological methods (such as ELISA and latex agglutination assays) have the core limitation that antibodies may cross-react with other microorganisms or host proteins, leading to false positive results. Furthermore, their sensitivity is insufficient in the early stages of infection or when the bacterial load is low, potentially resulting in false negatives. Taking PCR and its derivative technologies (qPCR, multiplex PCR) as an example, although they have high sensitivity and specificity, their detection procedures are extremely cumbersome. They require complex DNA extraction and purification of samples, and the entire process requires expensive, sophisticated instruments (PCR instruments, electrophoresis equipment, sequencers, etc.) and a strictly controlled molecular biology laboratory environment to prevent contamination. The operation must be performed by professional technicians.

[0004] Surface-enhanced Raman scattering (SERS) technology boasts significant advantages, including rapid response, high sensitivity, high specificity, rich "fingerprint-like" spectral information, no need for complex sample pretreatment, and ease of device miniaturization. SERS technology leverages the localized surface plasmon resonance effect generated by noble metal nanostructures (such as gold and silver nanoparticles) to greatly enhance the Raman signal on the target surface, thereby achieving highly sensitive detection of microbial surface components (such as cell wall polysaccharides, proteins, and metabolites). Its detection limit is as low as the single-cell level, far superior to microscopic examination and immunological methods, enabling accurate identification even in the early stages of contamination with extremely low bacterial loads.

[0005] SERS technology offers a simple operating procedure, typically eliminating the need for DNA extraction and purification or culture amplification, thus reducing sample pretreatment time and reagent consumption, and lowering the technical barrier for operators. Furthermore, SERS equipment can integrate a portable Raman spectrometer, supporting rapid on-site detection and real-time monitoring, making it highly suitable for field applications such as food processing lines and raw material acceptance stages, providing a powerful technical tool for the control of Candida albicans. Combining machine learning algorithms with SERS spectral data for pattern recognition and classification can further enable automated, high-throughput microbial identification and analysis, demonstrating promising application prospects and industrialization potential.

[0006] The preparation of ideal biocompatible SERS nanoprobes in existing technologies requires a multi-objective optimization process that simultaneously balances and optimizes multiple dimensions such as "signal strength", "signal stability", "targeting efficiency", "low background interference" and "bio-environmental adaptability". Summary of the Invention

[0007] The purpose of this invention is to provide a specific detection kit for Candida albicans and its application. This invention utilizes gold nanostars (AuNSs) to construct nanoprobes, using AuNSs as an enhancing substrate, and surface-modified with Raman probe molecules and Candida albicans-specific aptamers. The Raman probe molecules bind to the specific aptamers, enabling the differentiation of Candida albicans from other Candida species (such as Candida glabrata and Candida tropicalis) based on characteristic peaks, effectively avoiding false positives caused by cross-reactivity and significantly improving identification reliability. This kit provides rapid and specific detection of Candida albicans, and is simple to operate, time-efficient, and low-cost, solving the problems of complex sample pretreatment, low detection accuracy, long detection time, and cumbersome operation in existing Candida albicans detection technologies.

[0008] The technical solution adopted in this invention is:

[0009] This invention provides a Candida albicans-specific detection kit, the kit comprising a nanoprobe, wherein the nanoprobe is based on gold nanostars (AuNSs), and the substrate surface is modified with Raman probe molecules and a Candida albicans-specific aptamer; the nucleotide sequence of the Candida albicans-specific aptamer is shown in SEQ ID NO.1.

[0010] SEQ ID NO.1:

[0011] 5'-CGAAAGACCAACGCAGCCAAACUGAAGCCCCAGUCGCCCG-3'.

[0012] Furthermore, the kit also includes silicon wafers, referring to single-crystal silicon wafers with a resistivity in the range of 1–15 Ω. <100> P-doped silicon wafers.

[0013] Furthermore, the particle size of the gold nanostars is 50-100 nm.

[0014] Furthermore, the Raman probe molecule is 4-mercaptophenylboronic acid (4-MPBA).

[0015] Furthermore, the preparation method of the gold nanostar is as follows:

[0016] (1) Add the aqueous solution of chloroauric acid to a three-necked round-bottom flask and heat to boiling;

[0017] (2) Under vigorous stirring, add trisodium citrate aqueous solution to the solution in step (1) at a uniform rate, heat to 100°C under continuous stirring and maintain for 15-20 min until the color changes from pale yellow to wine red, cool to room temperature (25°C) to obtain gold nanoparticle (AuNPs) solution;

[0018] (3) Take 0.1-0.5mM (preferably 0.25 mM) chloroauric acid aqueous solution into a three-necked round bottom flask, and add hydrochloric acid, gold nanoparticle solution, silver nitrate aqueous solution and L-ascorbic acid aqueous solution in sequence under the conditions of room temperature and stirring at 800 rpm. React at 20-30℃ and 700-900 rpm until the solution color changes from pale yellow to gray-blue (preferably the reaction time is 30s); then add sodium dodecyl sulfate (SDS) aqueous solution, stir evenly to obtain gold nanostar solution (denoted as AuNSs), and store at 4℃ in the dark for later use.

[0019] Furthermore, in step (1), the concentration of the chloroauric acid aqueous solution is 0.5-5 mM (preferably 1 mM), and in step (2), the concentration of the trisodium citrate aqueous solution is 5 g / L-15 g / L (preferably 10 g / L). The volume ratio of the trisodium citrate aqueous solution to the chloroauric acid aqueous solution is 1:20-5:20 (preferably 3:20).

[0020] Further, in step (3), the concentration of hydrochloric acid is 0.5-5 M (preferably 1 M), the concentration of silver nitrate aqueous solution is 1-5 mM (preferably 2 mM), the concentration of L-ascorbic acid aqueous solution is 50-200 mM (preferably 100 mM), and the concentration of sodium dodecyl sulfate aqueous solution is 5-15 g / L (preferably 10 g / L); the volume ratio of chloroauric acid aqueous solution to hydrochloric acid, gold nanoparticle solution, silver nitrate aqueous solution, L-ascorbic acid aqueous solution, and sodium dodecyl sulfate aqueous solution is 100:(0.05-0.5):(0.5-5):(0.5-5):(0.1-1):(0.1-1), preferably 100:0.1:1:1:0.5:0.5.

[0021] Furthermore, the method for modifying the surface of gold nanostars with Raman probe molecules is as follows: at room temperature (25°C), an ethanol solution of Raman probe molecules is added to the gold nanostars, and an aqueous solution of sodium dodecyl sulfate is added. The mixture is allowed to stand at 20-30°C for 5-90 min (preferably 25°C, 60 min). After centrifugation (preferably 1500-7500 rpm, 15 min), the precipitate is dispersed in ultrapure water to obtain gold nanostars with surface-modified Raman probe molecules (denoted as AuNSs@4-MPBA).

[0022] Furthermore, the concentration of the Raman probe molecule ethanol solution is 0.08-250 mM (preferably 2 mM), the volume ratio of the Raman probe molecule ethanol solution to the gold core nanostar is 1:5-20, preferably 1:5; the volume ratio of the Raman probe molecule ethanol solution to the sodium dodecyl sulfate aqueous solution is 1:1-5, preferably 1:1; and the concentration of the sodium dodecyl sulfate aqueous solution is 5-15 g / L (preferably 10 g / L).

[0023] Furthermore, the preparation method of the nanoprobe is as follows:

[0024] An ultrapure aqueous solution of a Candida albicans-specific aptamer was reacted with gold nanostars with surface-modified Raman probe molecules at 20-30 °C for 5-90 min (preferably 15 min). The mixture was then separated by low-speed centrifugation (preferably 1500-7500 rpm for 15 min), and the precipitate was suspended in ultrapure water to obtain the nanoprobe (denoted as AuNSs@4-MPBA-Apt), which was stored at 4 °C in the dark.

[0025] Furthermore, the concentration of the ultrapure aqueous solution of the Candida albicans specific aptamer is 2.5-20 μM, preferably 7.5 μM; the volume ratio of the ultrapure aqueous solution of the Candida albicans specific aptamer to the gold nanostars with surface-modified Raman probe molecules is 1:5-15 (preferably 1:10).

[0026] The present invention also provides a detection method for the aforementioned Candida albicans specific detection kit, the method comprising the following steps:

[0027] 1) Vortex mix the bacterial solution to be tested with the nanoprobe and incubate at 37°C and 180 rpm for 5-120 min (preferably 15 min) to complete the biobinding reaction;

[0028] 2) Take the reaction solution from step 1) and drop it onto the surface of a silicon wafer, then allow it to dry at room temperature; the silicon wafer refers to a single crystal with a resistivity in the range of 1–15 Ω. <100> P-doped silicon wafers;

[0029] 3) SERS signals were acquired using a portable Raman spectrometer at an excitation wavelength of 785 nm and a laser power of 200 mW; the negative logarithm of the Candida albicans bacterial concentration (-logC, X-axis) and the probe molecule (4-MPBA at 1074 cm⁻¹) were established. -1 The semi-logarithmic calibration curve of the characteristic peak intensity (Y-axis) was obtained by using linear regression analysis to obtain the concentration of Candida albicans in the test bacterial solution.

[0030] Furthermore, in step 1), the volume ratio of the bacterial solution to the nanoprobe is 1:1-10, preferably 1:5.

[0031] Further, in step 3), the calibration curve was prepared as follows: Candida albicans ATCC 16404 bacterial suspensions of different concentrations were incubated with nanoprobes at 37℃ and 180 rpm for 5-120 min under constant temperature oscillation. The reaction solution was dropped onto the surface of a silicon wafer and dried at room temperature. SERS signals were acquired using a portable fiber optic probe Raman spectrometer. The calibration curve was plotted with the negative logarithm of the Candida albicans bacterial suspension concentration (-logC) as the abscissa and the intensity of the Raman probe molecular characteristic peak as the ordinate.

[0032] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0033] (1) The Candida albicans specific detection kit of the present invention uses AuNSs as SERS enhancement substrate and 4-MPBA as probe molecule, so that it has obvious characteristic peaks at the corresponding positions. It can be rapidly detected by collecting Raman spectra with portable Raman spectroscopy equipment without relying on traditional laboratory environment. It is suitable for a variety of on-site detection scenarios, significantly improving the flexibility and convenience of detection, and providing the possibility for rapid on-site detection.

[0034] (2) The Candida albicans-specific aptamer in the kit of the present invention can achieve [the following]: C. albicans The specific detection of this substance can be used for the analysis of trace amounts of Candida albicans in samples, with a detection limit of 1.14-1.21 CFU / mL.

[0035] (3) The kit of the present invention has high sensitivity and high recovery rate in complex food matrices (jam, milk and drinking water), and the linear correlation coefficient R² is greater than 0.99.

[0036] (4) The kit of the present invention has high reproducibility, uniformity and stability.

[0037] (5) Compared with traditional methods, the detection method of the kit of the present invention has the advantages of simple pretreatment process, short detection time and simple operation, which significantly reduces time and economic costs and is more suitable for rapid on-site detection of Candida albicans. Attached Figure Description

[0038] Figure 1 This is a schematic diagram illustrating the preparation of a Candida albicans detection kit.

[0039] Figure 2 This is a schematic diagram illustrating the operation of the Candida albicans detection kit.

[0040] Figure 3 The sequence and predicted secondary structure of the Candida albicans-specific aptamer (Ca-apt). (a) The randomized region of the aptamer is shown. (b) The predicted secondary structures of Ca-apt-1 and Ca-apt-12, which have the lowest folding energy, are shown. Nucleotides within the shaded regions correspond to the randomized regions of the aptamer.

[0041] Figure 4 Binding characteristics of Candida albicans-specific aptamers. (a) Aptamer screening for binding ability against Candida albicans strain ATCC90028, which was used as the screening target. Only Ca-apt-1 and Ca-apt-12 showed significantly higher binding ability than the negative control RNA. (b) The specificity of Ca-apt-1 and Ca-apt-12 was tested using the Candida albicans target strain ATCC 90028, clinically isolated Candida albicans strains, Saccharomyces cerevisiae, and Streptococcus mutans. (c) Aptamer-coupled immunosorbent assay (ALISA) using the two aptamers is shown and compared with Candida albicans antibody ELISA.

[0042] Figure 5The image shows the characteristic identification of AuNPs, AuNSs, and AuNSs@4-MPBA in Example 3; A represents the TEM image of AuNPs (20 nm); B represents the TEM image of AuNSs (50 nm); C represents the UV absorption spectrum and sample photograph of AuNPs and AuNSs; D represents the particle size distribution of AuNPs; E represents the particle size distribution of AuNSs; FH represents the XPS spectra of the C 1s orbital, Au4f orbital, and N 1s orbital of AuNSs@4-MPBA; I represents the XPS spectrum of AuNSs@4-MPBA.

[0043] Figure 6 The Raman spectrum is shown in Example 3, where I is 10. -9 The sample modified with M 4-MPBA ethanol solution, II was 10. -2 M 4-MPBA ethanol solution sample.

[0044] Figure 7 The image shows the zeta potential diagrams of AuNSs, AuNSs@4-MPBA, and AuNSs@4-MPBA-Apt in Example 3.

[0045] Figure 8 The images show the EDS spectra of AuNSs@4-MPBA (A) and AuNSs@4-MPBA-Apt (B) in Example 3.

[0046] Figure 9 The above are simulation diagrams of the electromagnetic field distribution of AuNSs in Example 3; A and B represent simulation diagrams of the electromagnetic field distribution of a single AuNSs in the XY and XZ planes, respectively; C and D represent simulation diagrams of the electromagnetic field distribution of two AuNSs in the XY and XZ planes, respectively.

[0047] Figure 10 Raman spectra of nanoprobes prepared by reacting gold nanostars with different concentrations of 4-MPBA in Example 4, and the relationship between 4-MPBA concentration and 1074 cm⁻¹. -1 A histogram showing the relationship between the intensity of characteristic peaks.

[0048] Figure 11 Raman spectra of nanoprobes prepared by reacting 4-MPBA with gold nanostars at different times in Example 4, and reaction times at 1074 cm⁻¹. -1 A histogram showing the relationship between the intensity of characteristic peaks.

[0049] Figure 12 Raman spectra of nanoprobes prepared by reacting gold nanostars with different aptamer concentrations in Example 4, and the relationship between aptamer concentration and 1074 cm⁻¹. -1 A histogram showing the relationship between the intensity of characteristic peaks.

[0050] Figure 13 Raman spectra of nanoprobes prepared by reacting the aptamer with gold nanostars at different times in Example 4, and reaction times at 1074 cm⁻¹. -1 A histogram showing the relationship between the intensity of characteristic peaks.

[0051] Figure 14 The Raman spectra of the nanoprobe incubated with Candida albicans culture for different times in Example 4, and the relationship between incubation time and 1074 cm⁻¹. -1 A histogram showing the relationship between the intensity of characteristic peaks.

[0052] Figure 15 This is a flowchart illustrating the usage of the Candida albicans detection kit.

[0053] Figure 16 Raman spectra and linear relationships between the intensity of characteristic peaks and the logarithm of Candida albicans concentration (LgC) after incubation of Candida albicans bacterial suspensions with nanoprobes at different concentrations.

[0054] Figure 17 A histogram showing the intensity of characteristic peaks for different strains detected by nanoprobes.

[0055] Figure 18 Raman spectra and RSD values ​​measured 10 times at the same site after Candida albicans bacterial suspension was incubated with nanoprobes.

[0056] Figure 19 Raman spectra and RSD values ​​at 10 different sites after Candida albicans bacterial suspension was incubated with nanoprobes.

[0057] Figure 20 The RSD values ​​are measured daily after Candida albicans bacterial suspension is incubated with nanoprobes and stored at 4°C for 30 days.

[0058] Figure 21 Raman spectra of drinking water samples after adding different concentrations of Candida albicans and the relationship between the logarithm of Candida albicans concentration and 1074 cm⁻¹ -1 The linear regression curve of the characteristic peak intensity.

[0059] Figure 22 Raman spectra of jam samples after adding different concentrations of Candida albicans and the relationship between the logarithm of Candida albicans concentration and 1074 cm⁻¹ -1 The linear regression curve of the characteristic peak intensity.

[0060] Figure 23 Raman spectra of milk samples after adding different concentrations of Candida albicans and the relationship between the logarithm of Candida albicans concentration and 1074 cm⁻¹ -1 The linear regression curve of the characteristic peak intensity. Detailed Implementation

[0061] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0062] The ultrapure water described in this embodiment of the invention refers to water with a resistivity of 18 MΩ. cm (25℃) of water.

[0063] The silicon wafer described in this embodiment of the invention refers to a single crystal with a resistivity in the range of 1–15 Ω and a size of 5 mm × 5 mm × 0.6 mm. <100> P-doped silicon wafers.

[0064] The room temperature mentioned in this invention refers to 25°C.

[0065] Example 1: Screening of aptamers

[0066] 1. Aptamer selection process

[0067] The SELEX screening process for aptamers first involves constructing a raw RNA library containing 1×10¹³ randomized central sequences of 40 nucleotides (nt) each, based on the target gene of Saccharomyces cerevisiae ATCC90028 (accession number MT193530.1). The central sequence is flanked by defined primer binding sites, with the sequence being: 5′-GGGAGUCGACCGACCAGAA [N40] UAUGUGCGUCUACAUCUAGACUCAU-3′ (84 nt in total).

[0068] The selection criteria for the SELEX process are shown in Table 1. Real-time quantitative PCR (q-PCR) was used to monitor the enrichment process of RNA aptamers. Specified amounts of RNA and Candida albicans ATCC 90028 were mixed in a 0.45 μm Millipore column, and the binding reaction was carried out in binding buffer (50 mmol / L HEPES buffer, pH 7.4, 10 mmol / L MgCl₂). The experiments were conducted using 100 mmol / L NaCl. To reduce non-specific RNA binding, 5 μg of baker's yeast tRNA was added as a competitor in each round of screening (see Table 1), for a total volume of 50 μl. Rounds 1 to 5 were incubated with gentle rotation at room temperature for 45 minutes, and from round 6 onwards, the incubation time was shortened to 30 minutes. After each round, cells were washed with binding buffer according to Table 1, and the bound RNA was eluted with 500 μl of elution buffer (8 mol / L urea, 5 mmol / L EDTA, pH 8.0). The eluted RNA was recovered by ethanol precipitation, amplified by q-PCR, and transcribed in vitro to produce RNA for the next round of screening. To enrich aptamers specific to Candida albicans, a reverse selection step was introduced in rounds 3, 5, and 10, using 5 × 10⁻⁶ NaCl. One cell line of *Saccharomyces cerevisiae* ATCC9763 was used. RNA that did not bind to *Saccharomyces cerevisiae* was then subjected to a binding reaction with *Candida albicans* using the method described above. After 11 rounds of selection, the RNA was cloned into a plasmid using the TOPO TA cloning kit and then transformed into One Shot® Top 10 chemically competent *E. coli* (Invitrogen, Carlsbad, CA). The plasmid containing the aptamer was purified using the QIAprep mini-extraction kit (Qiagen, Hilden, Germany) to obtain the RNA library.

[0069] In rounds 1 through 5 of the SELEX process, the enriched library components included both specific and non-specific binders. However, after the introduction of a reverse selection step in round 5, RNA retention gradually decreased from round 6 to round 10. We found that in round 11, the enrichment of RNA capable of binding to the target *Candida albicans* was significantly increased compared to the RNA from round 5 and the original library RNA. Therefore, the PCR products from round 11 were cloned to identify each aptamer. The random region sequences of the aptamers are shown below. Figure 3 As shown in Figure a, 12 aptamers were obtained. Except for Ca-apt-1 and Ca-apt-12 (identified four and twice respectively during the screening process), the remaining aptamers showed no shared sequences and were unique to each other. Ca-apt-1 and Ca-apt-12 were preferentially retained during the screening process, suggesting they may have high affinity for target cells. The predicted secondary structures were generated using RNAstructure 5.3, as shown below. Figure 3 As shown in b, the random region of Ca-apt-1 is 41 nucleotides (nt) long, longer than the original 40-nt RNA. Such changes can occur during reverse transcription or PCR steps in multiple rounds of the SELEX process.

[0070] Table 1. Screening scheme for Candida albicans-specific aptamers

[0071]

[0072] 2. Binding rate

[0073] The binding ability of the aptamer to the target Candida albicans was screened.

[0074] Approximately 10 6 One sample of *Candida albicans* ATCC90028 RNA and 100 pmol of library RNA (obtained in step 1) and 5 μg of *Bakeryus tRNA* were mixed in binding buffer to reduce nonspecific binding, with a total volume of 50 μl. After completing the binding, washing, and elution steps in step 1, the amount of RNA added and bound was quantified using q-PCR. The binding rate was calculated as (amount of bound RNA / amount of added RNA) × 100. Only the Ca-apt-1 and Ca-apt-12 aptamers showed significantly higher binding rates than the negative control RNA (a clone randomly selected from the original RNA library). Figure 4 (a). Therefore, these two aptamers were chosen for subsequent characterization.

[0075] To test the specificity of the aptamers and their binding ability to different targets, the binding of Ca-apt-1 and Ca-apt-12 aptamers to *Candida albicans*, *Saccharomyces cerevisiae* ATCC 9763, and *Streptococcus mutans* from ATCC or clinical samples was evaluated. The binding, washing, elution, and q-PCR quantification procedures were identical to those used in the screening experiments described above. "No binding activity" RNA clones were used as negative controls to confirm specificity. Relative binding affinity = (Percentage of aptamer binding to a specific microorganism) / (Percentage of non-binding RNA binding to the same microorganism).

[0076] The results are as follows Figure 4 As shown in Figure b, the Ca-apt-1 and Ca-apt-12 aptamers exhibited higher binding affinity to *Candida albicans* strain ATCC 90028 compared to RNA with no binding activity (i.e., those aptamer clones that did not show target binding). Furthermore, both aptamers could also recognize *Candida albicans* from clinical samples, although with lower binding rates. The Ca-apt-1 aptamer specifically bound to *Candida albicans*, while Ca-apt-12 cross-bound to *Saccharomyces cerevisiae* and *Streptococcus mutans*.

[0077] 3. Aptamer detection capability of Candida albicans

[0078] To confirm the above results and test whether the aptamer can be used for the specific detection of Candida albicans, the ability of the aptamer to detect Candida albicans was evaluated using an aptamer-coupled immunosorbent assay (ALISA). The specific steps are as follows:

[0079] (1) Dilute with 50 μl in coating buffer (50 mmol / L N) C A 96-well microplate (Iwaki, Tokyo, Japan) was coated with mouse anti-human Candida albicans monoclonal antibody (US Biological, Swampscott, Mass) at a concentration of 1 μg / ml and incubated overnight at 4°C. The plates were washed twice with PBST (50 mmol / L phosphate buffer containing 0.05% Tween 20, pH 7.2), and then 100 μl of PBST containing 1% BSA (Sigma, MA, USA) was added to each well. The plates were then blocked at room temperature (RT) for 90 minutes. (2) After washing with PBST, add 100 μl of PBST suspension (100 μl) of each of the following strains (5000 cells / ml): Candida albicans ATCC90028, clinically isolated Candida albicans, Saccharomyces cerevisiae ATCC 9763, and Streptococcus mutans ATCC700610 (three replicates for each strain) and 100 μl of binding buffer. Incubate with gentle shaking at room temperature for 1 hour, then wash the plate twice with PBST containing 0.1% Tween 20 to remove unbound targets. (3) Subsequently, add 100 μl (100 μg / ml) of binding buffer for biotinylated aptamers Ca-apt-1, Ca-apt-12, or unbound RNA (obtained in step 1) to each well and incubate at room temperature in the dark for 1 hour to allow binding. Wash three times with PBST to remove unbound material. (4) Finally, add 100 μl of streptavidin-horseradish peroxidase (HRP) conjugate solution diluted 1:1,000 to each well. After incubating on a shaking platform at room temperature for 30 minutes, wash the plate twice with PBST and perform color development using 100 μl of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt) as a substrate in the dark. The reaction is carried out by adding 100 μl of 0.25 M... S Termination. Absorbance was measured at 450 nm using a microplate reader (BioRad, USA), with washing buffer as a background control.

[0080] In step 3, (2) is changed to adding 100 μl of PBST suspension of Candida albicans ATCC90028 and 100 μl of binding buffer to each well at different concentrations (50 to 5,000 cells / ml, with three replicates for each concentration); (3) adding a biotinylated polyclonal anti-Candida albicans antibody for enzyme-linked immunosorbent assay (ELISA). Other procedures are the same.

[0081] See results Figure 4 All three ALISA methods, including c, Ca-apt-1, and Ca-apt-12, were able to detect Candida albicans at concentrations ranging from 50 to 5,000 cells / ml, and their performance was comparable to that of ELISA methods using Candida albicans-specific antibodies. Ultimately, Ca-apt-1 was selected as the aptamer for Candida albicans.

[0082] The aptamer nucleotide sequence (SEQ ID NO.1) of Candida albicans is as follows:

[0083] 5'-CGAAAGACCAACGCAGCCAAACUGAAGCCCCAGUCGCCCG-3'.

[0084] Example 2: Preparation of Nanoprobes

[0085] like Figure 1 As shown, the structure of the nanoprobe is based on gold nanostars (AuNSs), with Raman probe molecules and Candida albicans-specific aptamers modified on the substrate surface. The specific preparation process is as follows:

[0086] S1, the preparation method of gold nanostars is as follows:

[0087] (1) Take 40 mL of chloroauric acid (HAuCl4) An aqueous solution of 3H2O (1 mM) was placed in a three-necked round-bottom flask equipped with a reflux condenser, and then the solution was heated to boiling.

[0088] (2) Under vigorous stirring, quickly add 6 mL of 10 g / L trisodium citrate (C6H5Na3O7) to the solution from step (1). Prepare an aqueous solution of gold nanoparticles (AuNPs) with a particle size of 15-35 nm by maintaining the solution at 100°C for 15 minutes until the color changes from pale yellow to wine red. Then, allow it to cool to room temperature to obtain 46 mL of gold nanoparticle (AuNPs) solution.

[0089] (3) Take 200 mL of low-concentration chloroauric acid aqueous solution (0.25 mM) into a three-necked round-bottom flask, and add the following reagents in sequence: hydrochloric acid (200 μL, 1 M), gold nanoparticle solution (2 mL), silver nitrate (AgNO3) aqueous solution (2 mL, 2 mM), and L-ascorbic acid (L-AA) aqueous solution (1 mL, 100 mM). React at room temperature and under magnetic stirring at 800 rpm for 30 s until the color rapidly changes from pale yellow to grayish blue. After the reaction is complete, add 1 mL of sodium dodecyl sulfate aqueous solution (10 g / L) to the three-necked round-bottom flask while stirring, and stir evenly to obtain 206.2 mL of gold nanostar (AuNSs) solution with a particle size of 50-100 nm. Store at 4℃ in the dark for later use.

[0090] S2, the preparation method of modifying Raman probe molecules on the surface of gold nanostars is as follows:

[0091] At room temperature, 200 μL of 2 mM 4-MPBA ethanol solution was added to 1 mL of the gold nanostar solution prepared in step S1, and 200 μL of sodium dodecyl sulfate aqueous solution (10 g / L) was added. The mixture was mixed and allowed to stand for 1 h. After centrifugation (3500 rpm, 15 min), the excess 4-MPBA solution on the upper layer was removed, and 1 mL of ultrapure water was added to the precipitate for redispersement to obtain 1 mL of gold nanostar solution with surface-modified Raman probe molecules, denoted as AuNSs@4-MPBA.

[0092] S3, the preparation method of the nanoprobe is as follows:

[0093] 100 μL of Candida albicans-specific aptamer solution (7.5 μM, ultrapure water) was reacted with 1 mL of gold nanostar solution prepared in step S2 at room temperature for 15 min. After centrifugation (3500 rpm, 15 min), the excess aptamer solution in the upper layer was removed. 1 mL of ultrapure water was added to the precipitate for redispersibility, resulting in 1 mL of gold core nanostar solution with surface-modified Raman probe molecules and Candida albicans-specific aptamer, i.e., nanoprobe, denoted as AuNSs@4-MPBA-Apt, which was stored at 4℃.

[0094] The nucleotide sequence of the Candida albicans-specific aptamer (SEQ ID NO.1) is as follows:

[0095] 5'-CGAAAGACCAACGCAGCCAAACUGAAGCCCCAGUCGCCCG-3'.

[0096] Example 3: Characterization of Nanoprobe Structure

[0097] 1. Material morphology and composition analysis

[0098] The morphology and size of gold nanoparticles (AuNPs) and gold nanostars (AuNSs) were observed using transmission electron microscopy (TEM). The morphology of AuNSs@4-MPBA and AuNSs@4-MPBA-Apt was observed using cryo-scanning electron microscopy (SEM). The elemental composition and chemical valence state of 4-MPBA, AuNSs, and AuNSs@4-MPBA complexes were analyzed using X-ray photoelectron spectroscopy (XPS, Kratos AXIS Ultra DLD, Shimadzu, Japan). The UV absorption and particle size distribution of AuNPs and AuNSs were determined using a UV-Vis spectrophotometer (UV-1800, Shimadzu, Japan) and a nanoparticle size and Zeta potential analyzer (Malvern Zetasizer ZS90).

[0099] (1) Appearance and size

[0100] See results Figure 5 As shown, A and B are representative TEM images of the gold nanoparticles and gold nanostars prepared in Example 2. The gold nanoparticles are uniformly distributed spherical shapes with a size of about 20 nm (D). The gold nanostars show dendritic structures growing on their surface and have a size of about 60 nm (E).

[0101] (2) Ultraviolet absorption

[0102] UV-Vis spectra and sample colors of gold nanoparticles and gold nanostars are as follows: Figure 5 As shown in Figure C, gold nanoparticles exhibit a maximum absorption peak at 520 nm, while gold nanostars show a maximum absorption peak at 627.50 nm, indicating the successful synthesis of both gold nanoparticles and gold nanostars. Furthermore, the burgundy color of the gold nanoparticles and the grayish-blue color of the gold nanostars are easily visible to the naked eye.

[0103] (3) Elemental composition and chemical state

[0104] The elemental composition and chemical state of AuNSs, 4-MPBA, and AuNSs@4-MPBA were analyzed by X-ray photoelectron spectroscopy. Figure 5 The results showed that AuNSs mainly contained Ag and Au, which is consistent with the literature reports and further confirms the successful preparation of AuNSs. AuNSs@4-MPBA mainly contained Au, Ag, C, O, B, and S elements. High-resolution deconvolution analysis was performed on the Au, B, and S elements in the AuNSs@4-MPBA complex. Figure 5 (F, G, H). The deconvolution peaks at 83.68 eV and 87.38 eV correspond to Au 4f, respectively. 7 / 2and Au 4f 5 / 2 The deconvolution peak at 187.67 eV corresponds to BO. Furthermore, the appearance of the Au-S characteristic peak (165.02 eV) confirms the successful coupling of 4-MPBA and AuNSs. Other peaks correspond to CS (165.28 eV) and SO (169.37 eV, 170.67 eV), respectively. Based on these results, the successful synthesis of AuNSs@4-MPBA can be fully demonstrated.

[0105] 2. Enhancers of gold nanostars

[0106] At room temperature, take 200 μL of a concentration of 10 -9 mol·L -1 4-MPBA ethanol solution was added dropwise to 1 mL of the gold nanostar solution prepared by the method in Example 2. The reaction was allowed to proceed for 1 h. After centrifugation (3500 rpm, 15 min), the precipitate was redispersed in 1 mL of ultrapure water to obtain gold nanostars with surface-modified Raman probe molecules. These nanostars were then dropped onto a silicon wafer, air-dried at room temperature, and SERS was performed using a portable fiber optic probe Raman spectrometer (ATR3000, Xi'an, Shaanxi Province) with a 785 nm laser (500 mW power, 2 s acquisition time). The results are shown in the figure. Figure 6 Curve I. Under the same Raman test conditions, 200 μL of a concentration of 10... -2 mol·L -1 A 4-MPBA ethanol solution was dropped onto a silicon wafer for Raman spectroscopy detection. The results are shown in the figure. Figure 6 Curve II.

[0107] The enhancement factor of gold nanostars was calculated using the following formula:

[0108] EF=(I SERS ×C BULK ) / (I BULK ×C SERS )

[0109] Among them, I SERS It is 10 -9 mol·L -1 Gold nanostars prepared by adsorbing 4-MPBA on AuNSs at 1074 cm⁻¹ -1 Raman intensity at I BULK It is 10 -2 mol·L -1 1074 cm⁻¹ of 4-MPBA on silicon wafer -1 Raman intensity at point C BULK and C SERS The molecular concentration of 4-MPBA on the silicon wafer is 10. -2 mol·L-1 and 10 -9 mol·L -1 The final calculated enhancement factor is 1.66 × 10⁻⁶. 8 .

[0110] 3. Zeta potential

[0111] The zeta potentials of AuNSs, AuNSs@4-MPBA, and AuNSs@4-MPBA-Apt were determined using a nanoparticle size and zeta potential analyzer (Malvern Zetasizer ZS90). Figure 7 The average zeta potentials of AuNSs, AuNSs@4-MPBA, and AuNSs@4-MPBA-Apt were -41.9 mV, -56.9 mV, and -44.3 mV, respectively. After the addition of AuNSs to 4-MPBA, the ionization state of the phenylboronic acid group was highly pH-dependent; when pH > pKa (~8.5), the ionization state of the boronic acid group (-B(OH)) of 4-MPBA increased. It ionizes into negatively charged borate (-B(OH)₂)⁻. This significantly increases the surface negative charge density. The high curvature surface (star-shaped tip) of gold nanostars may enhance the adsorption capacity for 4-MPBA, forming a denser monolayer. Under alkaline conditions, the gold surface may adsorb more O2. ions (Au-O) The superposition of the negative charge of 4-MPBA with the aptamer may further reduce the zeta potential. After the addition of the aptamer, it may act as a "crosslinking agent" to connect multiple AuNSs, forming aggregates. Aggregation reduces the effective surface area of ​​the particles, lowers the surface charge density, and the long-chain structure of the aptamer may hinder the direct interaction between the surface charge and the solution, weakening electrophoretic mobility and potentially leading to a decrease in the absolute value of the zeta potential. These findings demonstrate the successful preparation of AuNSs@4-MPBA and AuNSs@4-MPBA-Apt.

[0112] 4. EDS spectrum

[0113] The EDS spectra of AuNSs@4-MPBA and AuNSs@4-MPBA-Apt were detected using an energy-dispersive surface scanning spectrometer (EDS mapping, accelerating voltage 200 kV). Figure 8 It can be seen that AuNSs@4-MPBA contains gold, silver, boron, oxygen, and sulfur, while AuNSs@4-MPBA-Apt contains gold, silver, boron, oxygen, phosphorus, and sulfur. This fully demonstrates the successful preparation of AuNSs@4-MPBA and AuNSs@4-MPBA-Apt.

[0114] 5. Simulation of AuNSs electric field distribution

[0115] Based on the morphology and size of gold nanostars observed by TEM, electromagnetic field distribution was simulated using finite-difference time-domain (FDTD) software to calculate the electric field distribution of the gold nanostar particles. The electric field was set to be polarized along the x-axis and incident on the AuNSs structure at an angle perpendicular to the z-axis. The mesh size of the simulation region was defined as 0.0046 nm × 0.0046 nm × 0.0046 nm. Perfectly matched layers were set at the boundaries in the x, y, and z directions to prevent non-physical scattering. The simulation convergence time was set to 2.39728 × 10⁻⁶. -6 fs ensures convergence of calculation results. This enables accurate simulation of the enhanced magnetic field region on the surface of gold nanostars, thereby obtaining more reliable simulation data of the electric field distribution. Figure 9 As shown, the SERS phenomenon is mainly due to the enhancement of the local electromagnetic field during LSPR resonance excitation. The mechanism of SERS enhancement in gold nanostars is explained by FDTD simulation. It can be seen that the electromagnetic field enhancement of gold nanostars mainly exists at the tip and corner of the gold nanostar.

[0116] Example 4: Synthesis and Parameter Optimization of Nanoprobes

[0117] 1. Optimization of 4-MPBA concentration

[0118] In step S2 of Example 2, the concentration of 4-MPBA was changed to 0.08 mM, 0.4 mM, 2 mM, 10 mM, 50 mM, and 250 mM, respectively, while other operations remained the same. Nanoprobes were prepared and dropped onto silicon wafers, then air-dried at room temperature. Raman spectroscopy was performed using a portable fiber optic probe Raman spectrometer (ATR3000, Xi'an, Shaanxi Province) with a 785 nm laser (500 mW power, 2 s acquisition time), and a 1074 cm⁻¹ plot was generated. -1 The relationship between the characteristic peak intensity and the 4-MPBA concentration is shown in the figure. Figure 10 The results showed that when the concentration of 4-MPBA increased from 0.08 mM to 2 mM, 4-MPBA at 1074 cm⁻¹... -1 The SERS signal at 1074 cm⁻¹ increased with increasing concentration; when the concentration of 4-MPBA increased from 2 mM to 250 mM, the SERS signal at 1074 cm⁻¹ increased. -1 The SERS signal at the concentration decreased with increasing concentration. Therefore, the optimal concentration was 200 μL of 2 mM 4-MPBA added to 1 mL of AuNSs.

[0119] Optimization of reaction time between 2-MPBA and gold nanostars

[0120] In Example 2, step S2, the reaction time of 1 h was changed to 5 min, 15 min, 30 min, 45 min, 60 min, and 90 min, respectively, while other operations remained the same. Nanotags were prepared for each change, and Raman spectroscopy was performed using the method in step 1. A 1074 cm⁻¹ plot was then generated. -1 The relationship between the intensity of the characteristic peak and the reaction time is shown in the figure. Figure 11 The results showed that when the reaction time was increased from 5 min to 60 min, 4-MPBA at 1074 cm⁻¹... -1 The peak intensity at the reaction time generally increases with time, gradually reaching saturation and adsorption equilibrium. Between 60 and 90 minutes, the peak intensity shows a slight decrease, possibly due to increased AuNSs aggregation caused by the prolonged reaction. Therefore, 60 minutes was selected as the optimal reaction time for 4-MPBA and AuNSs.

[0121] 3. Optimization of aptamer concentration

[0122] In step S3 of Example 2, the aptamer concentration was changed to 2.5, 5, 7.5, 10, 15, and 20 mM, respectively, while other operations remained the same. Nanoprobes were prepared for each, and Raman spectroscopy was performed using the method in step 1. A 1074 cm⁻¹ plot was then generated. -1 The relationship between the characteristic peak intensity and aptamer concentration is shown in the figure. Figure 12 The results showed that the aptamer concentration was 7.5 μmol·L⁻¹. -1 At that time, 4-MPBA was at 1074cm -1 The Raman spectral peak intensity is highest at a concentration greater than 7.5 μmol·L⁻¹. -1 At that time, 4-MPBA was at 1074 cm. -1 The Raman peak intensity gradually decreases at this point because excess aptamer affects 4-MPBA and thus induces AuNSs aggregation. Therefore, 7.5 μmol·L⁻¹ was chosen. -1 The optimal concentration of the aptamer.

[0123] 4. Optimization of reaction time between aptamers and surface-modified probe molecules in gold nanostars

[0124] In Example 2, step S3, the reaction time between the aptamer and the surface-modified Raman probe molecules in gold nanostars was changed from 15 min to 5 min, 15 min, 30 min, 45 min, 60 min, and 90 min, respectively, while other operations remained the same. Nanoprobes were prepared for each change, and Raman spectroscopy was performed using the method in step 1. A 1074 cm⁻¹ plot was then generated. -1 The relationship between the intensity of the characteristic peak and the reaction time is shown in the figure. Figure 13 The results showed that after 15 min of reaction, 4-MPBA was at 1074 cm⁻¹-1 The Raman peak intensity at 1074 cm⁻¹ increases; with prolonged reaction time, excessive aptamers may cover the already adsorbed 4-MPBA, causing the 4-MPBA peak intensity at 1074 cm⁻¹ to decrease. -1 The Raman peak intensity decreases at this point. Therefore, the optimal reaction time between the aptamer and AuNSs@4-MPBA is 15 min.

[0125] 5. Optimization of incubation time between nanoprobes and Candida albicans culture

[0126] 250 μL of the nanoprobe prepared by the method in Example 2 was mixed with 50 μL of the bacterial culture of Candida albicans (ATCC 90028) prepared by the method in Example 6 (10 4 (CFU / mL) was incubated at 37℃ and 180 rpm for 5 min, 15 min, 30 min, 45 min, 60 min, 90 min, and 120 min, respectively. Raman spectra were then detected using the method in step 1, and plotted at 1074 cm⁻¹. -1 The relationship between the intensity of the characteristic peak and the incubation time is shown in the figure. Figure 14 As shown, the results indicate that with increasing reaction time, 4-MPBA at 1074 cm⁻¹... -1 The Raman spectral peak intensity gradually increases, reaching its maximum at a reaction time of 15 min. Therefore, the optimal reaction time for Candida albicans with the SERS probe is 15 min.

[0127] Example 5: Detection method using a Candida albicans specific detection kit

[0128] like Figure 2 and Figure 15 As shown, the detection method of the Candida albicans specific detection kit includes the following steps:

[0129] (1) Mix 50 μL of the bacterial culture to be tested with 250 μL of the nanoprobe prepared by the method in Example 2 in a 2 mL centrifuge tube by vortexing and incubate at 37°C and 180 rpm for 15 min.

[0130] (2) Take 10 μL of the reaction solution and drop it onto the surface of the silicon wafer, then let it air dry at room temperature;

[0131] (3) SERS signal acquisition was performed using the portable fiber optic probe Raman spectrometer described in Example 4. The negative logarithm (-logC, X-axis) of the Candida albicans bacterial concentration and the relationship between the probe molecule and the concentration at 1074 cm⁻¹ were established according to Example 6. -1 The semi-logarithmic calibration curve of the characteristic peak intensity (Y-axis) Figure 14 The concentration of Candida albicans in the test bacterial solution was obtained by linear regression analysis.

[0132] Example 6: Sensitivity Evaluation of Nanoprobes

[0133] 50 μL of different concentrations (10 0 10 1 10 2 10 3 10 4 10 5 A CFU / mL Candida albicans (ATCC 90028) bacterial suspension was mixed with 250 μL of the nanoprobe prepared by the method in Example 2. The reaction and detection were performed using the method in Example 5. Raman spectroscopy was conducted at 1074 cm⁻¹. -1 The negative logarithmic relationship between Raman intensity and Candida albicans concentration is shown in the curve. Figure 16 As shown. Figure 16 It can be seen that 4-MPBA is at 1074cm -1 The Raman intensity at a certain point increases with increasing concentration of white rosary beads, exhibiting a good linear relationship with the logarithm of the white rosary bead concentration. The linear equation is y = 2373.80x + 7845.30 (R²). 2 =0.9949).

[0134] Preparation method of Candida albicans (ATCC 90028) bacterial suspension: Remove the Candida albicans glycerol cryovials from the -80℃ freezer and thaw rapidly in a 37℃ water bath. In a laminar flow hood, use a sterile inoculation loop to take a small amount of bacterial suspension and streak it onto a fresh YM solid medium plate. Invert the plate and incubate in a 28℃ incubator in the dark for 24-48 hours until single, smooth, milky-white, typical yeast-like colonies form. Pick a morphologically typical single colony from the above plate and streak it in a "Z" pattern onto 3 new YM solid medium plates to expand the culture. Incubate in a 28℃ incubator in the dark for 24-48 hours to obtain generation 1 Candida albicans. Prepare a generation 1 bacterial suspension using sterile physiological saline. 100 μL of the first-generation bacterial suspension was inoculated into YM solid medium and spread evenly using a disposable spreader. The culture was incubated at 28℃ for 24-48 hours to obtain second-generation spores. A second-generation bacterial suspension was prepared using sterile physiological saline. 100 μL of the second-generation bacterial suspension was then inoculated into YM solid medium and spread evenly using a disposable spreader. The culture was incubated at 28℃ for 24-48 hours to obtain third-generation spores. A third-generation bacterial suspension was prepared using sterile physiological saline. The third-generation bacterial suspension was centrifuged at 3000-5000 r / min for 5 minutes. The supernatant was carefully discarded, and the precipitate was redispersed in 1 mL of sterile physiological saline. The suspension was vortexed to ensure thorough mixing. Ten-fold serial dilutions of the bacterial suspension were prepared, and colony counting was performed using the plate count method at appropriate gradients to quantitatively analyze the original bacterial suspension. The entire quantitative process was performed in triplicate. The concentration of the bacterial suspension was adjusted to 1×10⁻⁶ using physiological saline.9 CFU / mL was used as the Candida albicans culture solution.

[0135] Preparation of YM solid culture medium: Weigh 21.0g of commercially available YM culture medium and 15g of agar powder, heat and dissolve in 1000mL of purified water, and autoclave at 121℃ for 15 minutes.

[0136] Example 7: Evaluation of the selectivity and specificity of nanoprobes

[0137] Aeromonas hydrophila ( Aeromonas hydrophila Preparation of ATCC 35654 bacterial suspension (denoted as AH): Using an inoculation loop, collect bacterial suspension or bacterial growth from a -80℃ glycerol storage tube or slant, streak onto NA medium, and incubate at 28-30℃ for 18-24 hours. Pick a single typical colony and inoculate it into a test tube containing 5-10 mL of nutrient broth, incubating at 28-30℃ with shaking at 200 rpm for 12-16 hours (to the late logarithmic growth stage). Serially dilute the above bacterial suspension using sterile physiological saline. Select a suitable dilution, and perform viable cell counting using the plate plating method (100 μL). Calculate the original bacterial suspension concentration from the plate colony count. All experiments are performed in triplicate. Adjust the bacterial suspension concentration to 1×10⁻⁶. 9 CFU / mL.

[0138] Legionella pneumophila subspecies ( Legionella pneumophila Preparation of ATCC 33152 bacterial suspension (denoted as LP): First, take the bacterial culture from the -80℃ storage tube and streak it onto a BCYE medium plate, then incubate at 37℃ with 2.5% C. Incubate in the environment for 3-5 days; select typical colonies and inoculate them onto fresh BCYE agar plates for secondary purification, culturing under the same conditions until abundant colonies are formed; scrape off the purified bacterial growth using a sterile inoculation loop and suspend it in 5 mL of BYE liquid medium to prepare a primary bacterial suspension; transfer 100 μL of the bacterial suspension to fresh BYE liquid medium and incubate at 37°C with shaking for 48-72 hours until the logarithmic growth phase; centrifuge the culture at 8000 rpm for 5 minutes, discard the supernatant, resuspend and wash with sterile physiological saline, repeat centrifugation once, and finally resuspend in an appropriate amount of physiological saline; perform preliminary quantification using a hemocytometer, and simultaneously perform 10-fold serial dilutions, selecting appropriate dilutions for plating on BCYE plates, incubating at 37°C for 5-7 days, and then count the colonies. Accurately calculate the stock solution concentration based on colony-forming units, and set up three parallel experiments throughout the process. Adjust the bacterial suspension to 1×10⁻⁶. 9 CFU / mL.

[0139] Acinetobacter baumannii ( Acinetobacter baumannii CICC 22933) bacterial suspension (denoted as AB), Shigella sonnei ( Shigella sonneiATCC 25931) bacterial suspension (denoted as SS), Bacillus pumilus ( Bacillus pumilus The preparation of ATCC 63202 bacterial suspension (denoted as BP) was the same as that of Aeromonas hydrophila, with the bacterial suspension adjusted to 1×10⁻⁶. 9 CFU / mL.

[0140] Candida albicans ( Candida albicans The preparation of the ATCC 90228 bacterial suspension (denoted as CA) was the same as in Example 5, except that the bacterial suspension was adjusted to 1×10⁻⁶. 9 CFU / mL.

[0141] Preparation of NA medium: Weigh 28.5.0g of commercially available NA medium, heat and dissolve it in 1000mL of purified water, and autoclave at 121℃ for 15 minutes.

[0142] BCYE medium preparation: Accurately weigh 10.0 g ACES buffer, 10.0 g yeast extract, 2.0 g activated carbon, 1.0 g α-ketoglutarate, and 15.0 g agar powder, dissolve in 900 mL deionized water and adjust the pH to 6.9, bring the volume to 990 mL, autoclave at 121 °C for 15 minutes, cool to 50-60 °C, and aseptically add 10 mL of a mixed aqueous solution containing 0.4 g L-cysteine ​​hydrochloride and 0.25 g soluble ferric pyrophosphate, which has been sterilized by filtration through a 0.22 μm filter membrane. Mix well, pour into plates, and store at 4 °C protected from light.

[0143] BYE medium: Weigh 10.0 g of ACES buffer, 10.0 g of yeast extract, 2.0 g of activated carbon, and 1.0 g of α-ketoglutarate. Dissolve them in 900 mL of purified water by heating and stirring. Adjust the pH to 6.90±0.05 with KOH solution. Make up the volume to 1000 mL with purified water. Autoclave at 121℃ for 15 minutes.

[0144] Nutrient broth culture medium: Weigh 13.0 g of commercially available nutrient broth culture medium powder, heat and dissolve it in 1000 mL of purified water, dispense it into containers, and autoclave it at 121℃ for 15 minutes.

[0145] Preparation of MIX1 mixed bacterial solution: Take 200 μL each of the above-mentioned Aeromonas hydrophila, Shigella sonnei, Bacillus pumilus, Legionella pneumophila subsp. and Acinetobacter baumannii bacterial solutions and mix them to obtain MIX1 mixed bacterial solution.

[0146] Preparation of MIX2 mixed bacterial solution: Take 200 μL each of the above-mentioned Aeromonas hydrophila, Shigella sonnei, Bacillus pumilus, Legionella pneumophila subsp., Acinetobacter baumannii and Candida albicans bacterial solutions and mix them to obtain MIX2 mixed bacterial solution.

[0147] The bacterial culture was tested using the method described in Example 5, and the result was 1074c. ¹ Raman signal intensity at point [location] is shown in [reference]. Figure 17 As shown.

[0148] Figure 17 The results showed that the Raman intensity of the Candida albicans-containing culture was significantly increased compared to the interfering bacteria, indicating that the SERS method has high specificity for Candida albicans. The Raman intensity of MIX2 containing Candida albicans was also much higher than that of MIX1, demonstrating the selectivity of the SERS method for Candida albicans. The high specificity and selectivity of this SERS method are due to the specific recognition and binding of the aptamer to the Candida albicans target.

[0149] Example 8: Evaluation of the reproducibility, uniformity, and stability of the nanoprobe

[0150] 1. Repeatability

[0151] (1) Take 50 μL of the Candida albicans (ATCC 90228) bacterial suspension prepared by the method in Example 6 (1.0 × 10⁻⁶) 9 The bacterial solution (CFU / mL) was mixed with 250 μL of the nanoprobe prepared by the method in Example 2 in a 1.5 mL centrifuge tube by vortexing and incubating at 37°C and 180 rpm for 15 min.

[0152] (2) Add 10 μL of the reaction solution to the surface of the silicon wafer and allow it to dry at room temperature; use the portable Raman probe spectrometer described in Example 4 to collect signals on the surface of the silicon wafer, and repeat the sampling at the same point 10 times. Calculate 1074c ¹The relative standard deviation (RSD) of the characteristic peak intensity is shown in the figure. Figure 18 To comprehensively evaluate probe performance.

[0153] Repeatability tests showed that the RSD values ​​of the peak intensity at the above displacements were all ≤5.2%, indicating good repeatability.

[0154] 2. Uniformity

[0155] In step 1 above, instead of sampling the same point 10 times, sample the different points 10 times. All other operations remain the same. See the results below. Figure 19 The results show that the RSD values ​​of the peak intensity at the above displacements are all ≤9.5%, indicating good uniformity.

[0156] 3. Stability

[0157] The reaction solution from step 1 (1) above was stored at 4°C for 30 days. Samples were taken daily and detected using the portable Raman probe spectrometer described in Example 4 (n=3). SERS spectra were collected, and the SERS peak (4-MPBA at 1074 cm⁻¹) was calculated.-1 The RSD of the intensity of the spectral peak at the location is shown in the figure. Figure 20 The results show that the peak intensity at the above displacement has an RSD value of 1.60%, indicating good stability.

[0158] Example 9: Determination of Candida albicans content in drinking water with pre-added ingredients

[0159] 1. Spike recovery rate

[0160] Centrifuge 10 mL of drinking water (4500 rpm, 15 min) to remove solid impurities. After autoclaving, add 10 mL of the supernatant to the Candida albicans culture prepared according to the method in Example 6 to achieve a final concentration of 7 × 10⁻⁶. 2 ~ 7×10 5 The concentration of Candida albicans was determined using the method described in Example 5, with CFU / mL as the test bacterial solution. The conventional plate count method was used for comparison. As shown in Table 2, the recovery rate of Candida albicans in the drinking water matrix reached 92.58%–102.86%.

[0161] Table 2 Recovery rates (n=3)

[0162]

[0163] 2. Sensitivity

[0164] To further evaluate the sensitivity of the nanoprobe in real samples, the final concentration of Candida albicans in step 1 above was changed to 7 × 10⁻⁶. 0 ~7×10 5 CFU / mL, all other procedures were the same, and Raman spectra were detected using the method in Example 5. 4-MPBA was plotted at 1074 c. The characteristic peak intensity at position ¹ is shown in the curve relating the logarithm of Candida albicans concentration (lg C). Figure 21 As shown. Experimental results show that 4-MPBA at 1074 c The intensity of the characteristic peak at ¹ increases in a dose-dependent manner with increasing Candida albicans concentration, showing a good linear relationship with the logarithm of concentration (lg C). In drinking water matrix, the linear equation is y = 2387.66x + 7741.04 (R²). 2 = 0.9998), LOD is 1.14 CFU / mL.

[0165] Example 10: Determination of the content of Candida albicans pre-added to jam

[0166] 1. Spike recovery rate

[0167] Commercially available jam was diluted with 9 times its volume of physiological saline, centrifuged (4500 rpm, 15 min) to remove solid impurities, and the supernatant was autoclaved. 10 mL of the supernatant was then added to the *Candida albicans* bacterial suspension prepared according to the method in Example 6 to achieve a final concentration of 1.75 × 10⁻⁶. 2 ~1.75×10 5 The concentration of Candida albicans was determined using the method described in Example 5, with CFU / mL as the test bacterial solution. The conventional plate count method was used for comparison. As shown in Table 2, the recovery rate of Candida albicans in the jam matrix reached 96.47%–104.70%.

[0168] 2. Sensitivity

[0169] To further evaluate the sensitivity of the nanoprobe in real samples, the final concentration of Candida albicans in step 1 above was changed to 1.75 × 10⁻⁶. 0 ~1.75×10 5 CFU / mL, all other procedures were the same, and Raman spectra were detected using the method in Example 5. 4-MPBA was plotted at 1074 °C. The characteristic peak intensity at position ¹ is shown in the curve relating the intensity to the logarithm of the bacterial concentration (lg C). Figure 22 As shown. The results indicate that 4-MPBA at 1074 c The intensity of the characteristic peak at position ¹ increases in a dose-dependent manner with increasing Candida albicans concentration, showing a good linear relationship with the logarithm of concentration (lg C). In the jam matrix, y = 2331.48x + 7895.87 (R 2 =0.9998), LOD is 1.21 CFU / mL.

[0170] Example 11: Determination of Candida albicans content in milk after pre-addition

[0171] 1. Spike recovery rate

[0172] Commercially available milk (pure milk, 13% solids content) was diluted with 9 times its volume of physiological saline, centrifuged (10000 rpm, 10 min) to remove solid impurities, and then autoclaved. 10 mL of the supernatant was added to the *Candida albicans* culture prepared according to the method in Example 6 to achieve a final concentration of 1.17 × 10⁻⁶. 2 ~1.17×10 5 The concentration of Candida albicans was determined using the method described in Example 5, with CFU / mL as the test bacterial solution. The conventional plate count method was used for comparison. As shown in Table 2, the recovery rate of Candida albicans in the milk matrix reached 91.45%–103.42%.

[0173] 2. Sensitivity

[0174] To further evaluate the sensitivity of the nanoprobe in real samples, the final concentration of Candida albicans in step 1 above was changed to 1.17 × 10⁻⁶. 0 ~1.17×10 5 CFU / mL, with other procedures remaining the same, Raman spectra were detected using the method described in Example 5, and 4-MPBA was plotted at 1074°C. The characteristic peak intensity at position ¹ is shown in the curve relating the intensity to the logarithm of the bacterial concentration (lg C). Figure 23 As shown. Experimental results show that 4-MPBA at 1074 c The intensity of the characteristic peak at position ¹ increases in a dose-dependent manner with increasing Candida albicans concentration, showing a good linear relationship with the logarithm of concentration (lg C). In the milk matrix, y = 2377.25x + 7822.24 (R 2 =0.9997 )LOD is 1.17CFU / mL.

[0175] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any partial changes to the formulation and process therein should be within the protection scope of the present invention.

Claims

1. A Candida albicans specific detection kit, characterized by, The kit includes a nanoprobe, which is based on gold nanostars and modified on the substrate surface with Raman probe molecules and Candida albicans-specific aptamers; the nucleotide sequence of the Candida albicans-specific aptamer is shown in SEQ ID NO.

1.

2. The Candida albicans-specific test kit as set forth in claim 1, characterized by, The gold nanostars have a particle size of 50-100 nm, and the Raman probe molecule is 4-mercaptophenylboronic acid.

3. The Candida albicans-specific test kit as set forth in claim 1, characterized by, The method for preparing the gold nanostars is as follows: (1) Add the aqueous solution of chloroauric acid to a three-necked round-bottom flask and heat to boiling; (2) Under vigorous stirring, add trisodium citrate aqueous solution to the solution in step (1) at a uniform rate, heat to 100°C and maintain for 15-20 min under continuous stirring, cool to room temperature to obtain gold nanoparticle solution; (3) Take 0.1-0.5mM chloroauric acid aqueous solution into a three-necked round bottom flask, and add hydrochloric acid, gold nanoparticle solution, silver nitrate aqueous solution and L-ascorbic acid aqueous solution in sequence under room temperature and stirring conditions. React at 20-30℃ and 700-900 rpm until the solution color turns gray-blue; then add sodium dodecyl sulfate aqueous solution and stir evenly to obtain gold nanostars.

4. The Candida albicans-specific test kit as claimed in claim 3, wherein the Candida albicans-specific probe is a polynucleotide probe having a nucleotide sequence of SEQ ID NO:

1. In step (1), the concentration of chloroauric acid aqueous solution is 0.5-5 mM; in step (2), the concentration of trisodium citrate aqueous solution is 5-15 g / L; the volume ratio of trisodium citrate aqueous solution to chloroauric acid aqueous solution is 1-5:20; in step (3), the concentration of hydrochloric acid is 0.5-5 M, the concentration of silver nitrate aqueous solution is 1-5 mM, the concentration of L-ascorbic acid aqueous solution is 50-200 mM, and the concentration of sodium dodecyl sulfate aqueous solution is 5-15 g / L; the volume ratio of chloroauric acid aqueous solution to hydrochloric acid, gold nanoparticle solution, silver nitrate aqueous solution, L-ascorbic acid aqueous solution, and sodium dodecyl sulfate aqueous solution is 100:0.05-0.5:0.5-5:0.5-5:0.1-1:0.1-1.

5. The Candida albicans specific detection kit as described in claim 1, characterized in that, The method for modifying Raman probe molecules on the surface of gold nanostars is as follows: At room temperature, an ethanol solution of Raman probe molecules is added to the gold nanostars, and an aqueous solution of sodium dodecyl sulfate is added. The mixture is allowed to stand at 20-30 °C for 5-90 min. After centrifugation, the precipitate is dispersed in ultrapure water to obtain gold nanostars with Raman probe molecules modified on the surface.

6. The Candida albicans specific detection kit as described in claim 5, characterized in that, The concentration of the Raman probe molecule ethanol solution is 0.08-250 mM, and the volume ratio of the Raman probe molecule ethanol solution to gold nanostars is 1:5-20; the volume ratio of the Raman probe molecule ethanol solution to sodium dodecyl sulfate aqueous solution is 1:1-5, and the concentration of the sodium dodecyl sulfate aqueous solution is 5-15 g / L.

7. The Candida albicans specific detection kit as described in claim 1, characterized in that, The preparation method of the nanoprobe is as follows: The ultrapure aqueous solution of the Candida albicans specific aptamer was reacted with gold nanostars with surface-modified Raman probe molecules at 20-30℃ for 5-90 min. The precipitate was then separated by centrifugation and suspended in ultrapure water to obtain the nanoprobe.

8. The Candida albicans specific detection kit as described in claim 7, characterized in that, The concentration of the ultrapure aqueous solution of the Candida albicans specific aptamer is 2.5-20 μM; the volume ratio of the ultrapure aqueous solution of the Candida albicans specific aptamer to the gold nanostars with surface-modified Raman probe molecules is 1:5-15.

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