CN115236057A

CN115236057A - Method for simultaneously detecting three pathogenic bacteria based on lectin and aptamer dual recognition

Info

Publication number: CN115236057A
Application number: CN202210705323.5A
Authority: CN
Inventors: 关明; 米芳; 王莹; 陈国通; 耿鹏飞; 胡存明
Original assignee: Xinjiang Normal University
Current assignee: Xinjiang Normal University
Priority date: 2022-06-21
Filing date: 2022-06-21
Publication date: 2022-10-25

Abstract

The invention discloses a method for simultaneously detecting three pathogenic bacteria based on double recognition of agglutinin and aptamer. The method comprises the following steps: incubating the lectin magnetic nanoparticles and three different types of pathogenic bacteria together for 30min, and washing by PBS under the action of an external magnetic field to remove the unbound pathogenic bacteria; preparing three SERS probes with different types and functionalized by aptamers, incubating the SERS probes with lectin magnetic nanoparticles for capturing pathogenic bacteria for 30min, washing the SERS probes with PBS for 3 times to remove the unbound SERS probes, performing magnetic separation, redissolving the original volume, and performing detection analysis by using a portable Raman spectrometer. The invention selects three different types of aptamer functionalized SERS probes which are mutually free of interference to be respectively specifically combined with three pathogenic bacteria and incubated with lectin functionalized magnetic nanoparticles with wide pathogenic bacteria capturing functions to form a sandwich structure, thereby solving the problem of simultaneous quantitative detection of multiple pathogenic bacteria in the current complex sample.

Description

Method for simultaneously detecting three pathogenic bacteria based on lectin and aptamer dual recognition

Technical Field

The invention belongs to the technical field of food detection, and particularly relates to a method for simultaneously detecting three pathogenic bacteria based on lectin and aptamer dual recognition.

Background

The Surface-enhanced Raman Scattering (SERS) technology is used as a biological fingerprint identification technology, and provides a new research visual angle for rapidly and accurately detecting pathogenic bacteria in recent years. In 1974, fleischmann et al, carried out raman spectroscopy of pyridine, and found that when pyridine was adsorbed on a silver electrode having a rough surface, a raman spectrum of a pyridine molecule having a strong characteristic peak and an excellent peak shape was observed. In 1977, systematic calculation of the Van Duyne group based on Fleischmann et al found that when unit pyridine molecules were adsorbed on the surface of a rough silver electrode, the measured Raman spectrum signal intensity was about 10 times stronger than that obtained by directly detecting the pyridine molecules ⁵ ～10 ⁶ And (4) doubling. The detection of pathogenic bacteria is mainly divided into marked detection and unmarked detection, the unmarked detection is mainly to observe the fingerprint of bacteria through the combination of the pathogenic bacteria and nano particles, the characteristics of difficult regulation and control and poor repeatability exist, in addition, the fingerprint difference among the pathogenic bacteria is very weak, and some pathogenic bacteria are difficult to directly distinguish according to the SERS fingerprint of the pathogenic bacteria, so the difficulty of field detection is increased.

Disclosure of Invention

The invention aims to provide a method for simultaneously detecting three pathogenic bacteria based on double recognition of lectin and aptamer, which solves the problems in the prior art by specifically combining three SERS probes with high-strength SERS signals and without mutual interference with the pathogenic bacteria and forming a sandwich structure together with the pathogenic bacteria by lectin functionalized magnetic nanoparticles with a wide pathogenic bacteria capturing function.

In order to solve the technical problems, the invention is realized by the following technical scheme:

the invention relates to a method for simultaneously detecting three pathogenic bacteria based on double recognition of agglutinin and aptamer, which comprises the following steps:

stp1, incubating magnetic nanoparticles of the lectin with three different types of pathogenic bacteria for 1h, and washing with PBS under the action of an external magnetic field to remove unbound pathogenic bacteria;

stp2, preparing aptamer-functionalized SERS probes of three different types, and then incubating the SERS probes with lectin magnetic nanoparticles captured with pathogenic bacteria for 30min to form a sandwich structure for simultaneous quantitative detection of the three different types of pathogenic bacteria;

the three SERS probes comprise an Au-4MPBA @ Ag-P1 SERS probe, an Au @ DTNB @ Ag-P2SERS probe and an Au @ Ag @ PB @ Ag-P3 SERS probe;

stp3, washing for 3 times by using PBS to remove the unbound SERS probe, redissolving the original volume after magnetic separation, and analyzing by using a portable Raman spectrometer; in Stp3, recording a Raman spectrum by adopting a 785nm laser with 200mW power, wherein the integration time is 500ms, the accumulated Raman spectrum recorded for 3 times is an average value of 5 repeated measurements, and all Raman spectra are subjected to baseline correction;

the PBS concentration in Stp1 and Stp3 was 0.1M, pH =7.4; the preparation of the 0.1M PBS solution with pH =7.4 specifically was: 7.8g of sodium dihydrogen phosphate (NaH) was accurately weighed ₂ PO ₄ ) 475mL of deionized water is added, stirred and dissolved sufficiently, then 2.0g of NaOH is weighed to prepare 1.0M NaOH solution, the pH value of the solution is adjusted to 7.4, the solution is transferred to a 500mL volumetric flask, deionized water is added to 500mL, and the solution is mixed sufficiently for standby.

Further, the three pathogenic bacteria are respectively escherichia coli (E.coil), staphylococcus aureus (S.aureus) and pseudomonas aeruginosa (P.aeruginosa);

the three aptamers are respectively escherichia coli aptamers (P1: 5'-COOH-GCA ATG GTA CGG TAC TTC CTC GGC ACG TTC TCA GTA GCG CTC GGT CAT CCC ACA GCT ACG TCA AAA GTG CAC GCT ACT TTG CTA A-3');

staphylococcus aureus aptamer (P2: 5'-COOH-ATC CGT CAC ACC TGC TCT ACTGGC CGG CTC AGC ATG ACT AAG GAA GTT ATG TGG TGT TGG CTC CCG TAT-3');

pseudomonas aeruginosa aptamer (P3: 5'-SH-CCC CCG TTG CTT TCG CTT TTC CTT TCG CTT TTG TTC GTT TCG TCC CTG CTT CCT TTC TTG-3');

the preparation method of the lectin magnetic nanoparticles comprises the following steps:

stp01, amino-modified magnetic nanoparticle (Fe) ₃ O ₄ @SiO ₂ -NH ₂ ) The preparation of (1):

stp11, 2.1g FeCl ₃ ·6H ₂ Adding O, 4g of anhydrous NaAc and 1g of polyethylene glycol into 60mL of ethylene glycol, stirring to completely dissolve, placing into a 100mL high-pressure reaction kettle with a polytetrafluoroethylene lining, and reacting for 8 hours at the temperature of 195-198 ℃;

stp12, cooling, washing with water and ethanol for several times, and drying at 60 deg.C in vacuum drying oven to obtain Fe ₃ O ₄ NPs；

Stp13, 200gFe ₃ O ₄ NPs are added into 60mL of absolute ethyl alcohol and 15mL of water, and are fully dispersed by ultrasonic;

stp14, adding 3mL of ammonia water and 0.4mL of TEOS in sequence, stirring for 1.5h, finally washing with water and ethanol for several times in a crossed manner, and drying at 60 ℃ for 6h to obtain silicon dioxide coated magnetic nanoparticles Fe3O4@ SiO2;

stp15 and Fe obtained above ₃ O ₄ @SiO ₂ Dispersing into 100mL of absolute ethanol containing 5mL of APTES, and refluxing at 60 ℃ for 12h while mechanically stirring the mixture; finally, collecting a magnet, washing the magnet for a plurality of times by using water and ethanol, and drying the magnet in vacuum to obtain an amino modified magnetic material, wherein the product is marked as AMNPs;

stp02, boric acid modified Fe ₃ O ₄ @SiO ₂ The preparation of (1):

dissolving 100mg of AMNPs in 50mL of methanol, adding 200mg of FPBA and 250mg of NaBH3CN, carrying out ultrasonic dispersion, stirring for 24h at room temperature, carrying out cross washing on the product for a plurality of times by using water and methanol, and carrying out vacuum drying overnight at 60 ℃, thus obtaining the product called FPBA-AMNPs;

stp03, dissolving lectin (ConA) in PBS with concentration of 0.1M and pH of 7.4 to prepare stock solution with concentration of 5mg/100 mL; adding 100mg of FPBA-AMNPs into the stock solution, mechanically stirring at room temperature for 15min, and storing at 4 deg.C.

The preparation of the three SERS probes comprises the following steps:

first, 100mL 0.01% of HAuCl is taken ₄ ·4H ₂ Placing O (aq) in a three-neck flask, placing in a reflux device, magnetically stirring and heating to boil; then, adding 1mL of 3% trisodium citrate solution rapidly, and continuing stirring for 30min, wherein the solution is wine red; finally, the obtained gold nanoparticles (AuNPs) are naturally cooled to room temperature, filled in a clean vessel and stored at 4 ℃ before use.

The preparation of the Au-4MPBA @ Ag-P1 SERS probe also comprises the steps of adding 1mL of 1mM 4-MPBA into 10mL of the prepared AuNPs; magnetically stirring for reaction for 15min, centrifuging, removing supernatant, and dissolving again with deionized water to obtain Au-4 MPBA; then adding 1mL20mM AgNO3 and 1mL0.02g/mL ascorbic acid in sequence; and after the reaction is carried out for 10min by magnetic stirring, removing unreacted excessive materials by centrifugation and re-dissolving the excessive materials in deionized water to obtain Au-4MPBA @ AgNPS, and oscillating and incubating the Au-4MPBA @ AgNPS and the aptamer P1 for 10min to obtain the aptamer functionalized Au-4MPBA @ Ag-P1NPsSERS probe.

The preparation of Au @ DTNB @ Ag-P2SERS probe also included adding 1mL of 10mM DTNB to 10mL of the prepared AuNPs; magnetically stirring for reaction for 15min, centrifuging, removing supernatant, and dissolving again with deionized water to obtain Au-DTNB; then sequentially adding 1mL of 20mM AgNO3 and 1mL0.02g/mL ascorbic acid; and after the reaction is carried out for 10min by magnetic stirring, removing unreacted excessive materials by centrifugation, re-dissolving the excessive materials in deionized water to obtain Au-DTNB @ AgNPS, and carrying out co-oscillation incubation with the aptamer P2 for 10min to obtain the aptamer functionalized Au-4 DTNB @ Ag-P2NPsSERS probe.

The preparation of the Au @ Ag @ PB @ Ag-P3 SERS probe further comprises the step of adding 1mL0.1g/mL of ascorbic acid into 10mL of prepared AuNPs; after the reaction is carried out for 15min by magnetic stirring, 1mL of 20mM AgNO3 is added to continue the magnetic stirring for 15min, and Au @ Ag nano particles are obtained; then 1mL of 1mM FeCl3.6H2O and 1mL of 1mM K are added in this order ₄ [Fe(CN) ₆ ]·3H ₂ O, after magnetically stirring and reacting for 10min, separatingThe supernatant is removed by the heart, 200 mu L of 10mM AgNO3 is added for magnetic stirring for 10min, the excess material which is not reacted is removed by centrifugation and is dissolved in deionized water again to obtain Au @ Ag @ PB @ AgNPs, and the Au @ Ag @ PB @ Ag-P3NPsSERS probe functionalized by oscillating and incubating with the aptamer P3 for 10min is obtained.

In our experiments, shock frozen E.coli, staphylococcus aureus and Pseudomonas aeruginosa were used as models. Bacterial cells were cultured on Nutrient Broth (NB) solid medium at 37 ℃ for 24 hours. Then, the bacteria were washed with 5mL of sterile water, and 5mL of bacterial suspension was collected by centrifugation at 4000rpm at 4 ℃ and then washed three times with PBS. Finally, the bacterial cells were diluted to the desired concentration with PBS buffer and measured using an ultraviolet spectrophotometer OD 600.

The laser power of the portable surface enhanced Raman spectrometer is 200mw, the wavelength of the excitation light is 785nm, and the scanning spectral range is 400-2500cm ^-1 The integration time was 500ms, and the mean spectra were taken after 3 scans of each sample and baseline corrected. 2mL of ConA @ FPBA-AMNPs and 1mL of bacterial suspension (varying in concentration for experimental purposes) were added to a 5mL centrifuge tube. The mixture was then incubated for 1 hour with shaking. Thereafter, the bacteria/ConA @ FPBA-AMNPs complex was isolated under magnetic field and washed with PBS to remove unbound bacteria. Subsequently, 1mL of different SERS probes (Au-MPBA @ Ag-P1, au-DTNB @ Ag-P2, au @ Ag @ PB @ Ag-P3) were added to the magnetically separated bacteria/ConA @ FPBA-AMNPs complex. The obtained SERS probe/bacteria/ConA @ FPBA-AMNPs sandwich structure is subjected to magnetic separation, washed 3 times by PBS buffer solution, and then redissolved by 1mL PBS and subjected to quantitative detection of pathogenic bacteria by a portable Raman spectrometer.

The invention has the following beneficial effects:

the invention adopts three SERS probes which have high-strength SERS signals and do not interfere with each other to perform specific combination with pathogenic bacteria, and forms a sandwich structure together with the pathogenic bacteria through magnetic nanoparticles which have wide lectin capturing functions with the pathogenic bacteria; the method is simple in preparation process, good in stability and extremely low in detection limit, provides basic research for on-site real-time detection of pathogenic bacteria in SERS detection, and provides technical support for simultaneously detecting three different types of pathogenic bacteria from a complex substrate by selecting three mutually-noninterference SERS probes.

1. The invention aims to specifically combine target pathogenic bacteria, simultaneously and quantitatively detect three different types of pathogenic bacteria in a complex sample through strong Raman signals, and capture the pathogenic bacteria by a magnetic material with lectin function, thereby improving the enrichment and separation efficiency and completing the whole detection process within 1 h.

Of course, it is not necessary for any product in which the invention is practiced to achieve all of the above-described advantages at the same time.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic diagram of SERS detection principle of three food-borne pathogenic bacteria based on dual recognition ligands;

FIG. 2 is a schematic diagram of the preparation principle of three SERS probes Au-MPBA @ Ag-P1, au-DTNB @ Ag-P2 and Au @ Ag-PB @ Ag-P3;

FIG. 3 is a representation of Au-MPBA @ AgNPs, au-DTNB @ AgNPs;

FIG. 4 (A) is a TEM image of AuNPs;

FIG. 4 (B) is a TEM image of Au @ AgNPs;

FIG. 4 (C) is a TEM image of Au @ Ag-PBNPs;

FIG. 4 (D) is a graph showing the distribution of the AuNPs;

FIG. 4 (E) is a graph showing the distribution of particle size distribution of Au @ AgNPs;

FIG. 4 (F) is a graph showing the particle size distribution of Au @ Ag-PB NPs;

FIG. 4 (G) is a TEM image of Au @ Ag-PB @ Ag NPs;

FIG. 4 (H) is the HAADF-STEM image of Au @ Ag-PB @ Ag NPs, au (purple), ag (green), fe (yellow), K (red);

FIG. 4 (I) is a particle size distribution diagram of Au @ Ag-PB @ Ag NPs;

FIG. 5A (a) shows Fe ₃ O ₄ The TEM of (4);

FIG. 5A (b) Fe ₃ O ₄ @SiO ₂ The TEM of (4);

FIG. 5A (c) shows Fe ₃ O ₄ SEM of (2);

FIG. 5A (d) is Fe ₃ O ₄ @SiO ₂ SEM of (2);

FIG. 5B is an FT-IR spectrum of Fe3O4, fe3O4@ SiO2, AMNP, FPBA-AMNPs;

FIG. 5C is Fe ₃ O ₄ 、Fe ₃ O ₄ @SiO ₂ XRD spectra of FPBA-AMNPs;

FIG. 5D shows Fe ₃ O ₄ And XPS spectra of FPBA-AMNPs;

FIG. 6 (A) is a hysteresis curve of Fe3O4, fe3O4@ SiO2, FPBA-AMNPs;

FIG. 6 (B) is adsorption equilibrium and model fitting of FPBA-AMNPs to ConA;

FIG. 6 (C) is Scatchard fit data of FPBA-AMNPs to ConA binding;

FIG. 6 (D) is the regeneration capacity of FPBA-AMNPs for ConA adsorption;

FIG. 7 (A) is the Raman spectrum of Au-DTNB @ Ag, au-MPBA @ Ag, au @ Ag-PB @ Ag and the mixture of three SERS probes;

FIG. 7 (B) is the Raman spectrum of three SERS probes of Au-DTNB @ Ag, au-MPBA @ Ag and Au @ Ag-PB @ Ag mixed for detecting three mixed bacteria of Staphylococcus aureus, escherichia coli and Pseudomonas aeruginosa with different concentrations;

FIG. 7 (C) is a SERS signal comparison of mixed detection of Staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, listeria monocytogenes, salmonella typhi, and Bogder shigelidi;

FIG. 7 (D) is 1078cm ^-1 (ii) raman peak intensity versus staphylococcus aureus concentration (n = 5);

FIG. 7 (E) is 1334cm ^-1 (ii) raman peak intensity versus e.coli concentration (n = 5);

FIG. 7 (F) is 2081cm ^-1 (iii) raman peak intensity versus pseudomonas aeruginosa concentration (n = 5).

Detailed Description

A method for simultaneously detecting three pathogenic bacteria based on double recognition of lectin and aptamer comprises the following steps:

stp1, incubating magnetic nanoparticles of the lectin with three different types of pathogenic bacteria for 1h, and washing with PBS (phosphate buffer solution) with the concentration of 0.1M and the pH =7.4 under the action of an external magnetic field to remove unbound pathogenic bacteria; the effective enrichment and separation of pathogenic bacteria are realized by the combination of the lectin magnetic nanoparticles and the pathogenic bacteria; the preparation of the 0.1M PBS solution with pH =7.4 was specifically: 7.8g of sodium dihydrogen phosphate (NaH) was accurately weighed ₂ PO ₄ ) 475mL of deionized water is added, stirred and dissolved sufficiently, then 2.0g of NaOH is weighed to prepare 1.0M NaOH solution, the pH of the solution is adjusted to 7.4, the solution is transferred to a 500mL volumetric flask, deionized water is added to 500mL, and the mixture is mixed sufficiently for standby.

The three pathogenic bacteria are respectively escherichia coli (E.coil), staphylococcus aureus (S.aureus) and pseudomonas aeruginosa (P.aeruginosa);

the three aptamers were:

escherichia coli aptamer (5 '-COOH-GCA ATG GTA CGG TAC TTC CTC GGC ACG TTC TCA GTA GCG CTC GCT GGT CAT CCC ACA GCT ACG TCA AAA GTG CAC GCT ACT TTG CTA A-3');

staphylococcus aureus aptamer (5 '-COOH-ATC CGT CAC ACC TGC TCT ACTGGC CGG CTC AGC ATG ACT AAG GAA GTT ATG TGG TGT TGG CTC CCG TAT-3');

pseudomonas aeruginosa aptamer (5 '-SH-CCC CCG TTG CTT TCG CTT TTC CTT TCG CTT TTG TTC GTT TCG TCC CTG CTT CCT TTC TTG-3');

stp3, washing with PBS (phosphate buffer solution) with the concentration of 0.1M and the pH =7.4 for 3 times to remove the unbound SERS probe, re-dissolving the original volume after magnetic separation, recording a Raman spectrum by adopting 785nm laser with the power of 200mW, wherein the integration time is 500ms, and accumulating for 3 times; the recorded raman spectra are the average of 5 replicates and all raman spectra are subjected to baseline correction analysis.

In our experiments, shock frozen E.coli, S.aureus and P.aeruginosa were used as models. Bacterial cells were cultured on Nutrient Broth (NB) solid medium at 37 ℃ for 24 hours. Then, the bacteria were washed with 5mL of sterile water, and 5mL of bacterial suspension was collected by centrifugation at 4000rpm at 4 ℃ and then washed three times with PBS. Finally, the bacterial cells were diluted to the desired concentration with PBS buffer and assayed using an ultraviolet spectrophotometer OD 600.

The laser power of the portable surface enhanced Raman spectrometer is 200mw, the wavelength of the excitation light is 785nm, and the scanning spectral range is 400-2500cm ^-1 The integration time was 500ms, and the mean spectra were taken after 3 scans of each sample and baseline corrected. 2mL ConA @ FPBA-AMNPs and 1mL bacterial suspension (different concentrations for different purposes of the experiment) were added to a 5mL centrifuge tube. The mixture was then incubated for 1 hour with shaking. Thereafter, the bacteria/ConA @ FPBA-AMNPs complex was separated under magnetic field and washed with PBS to remove unbound bacteria. Subsequently, 1mL of different SERS probes (Au-MPBA @ Ag-P1, au-DTNB @ Ag-P2, au @ Ag @ PB @ Ag-P3) were added to the magnetically separated bacteria/ConA @ FPBA-AMNPs complex described above. The obtained SERS probe/bacteria/ConA @ FPBA-AMNPs sandwich structure is subjected to magnetic separation, washed 3 times by PBS buffer solution, and then redissolved by 1mL PBS and subjected to quantitative detection of pathogenic bacteria by a portable Raman spectrometer.

stp11, 2.1g FeCl ₃ ·6H ₂ O, 4g of anhydrous NaAc and 1g of polyethyleneAdding glycol into 60mL of ethylene glycol, stirring to completely dissolve the glycol, placing the glycol into a 100mL high-pressure reaction kettle with a polytetrafluoroethylene lining, and reacting for 8 hours at the temperature of 195-198 ℃;

stp14, adding 3mL of ammonia water and 0.4mL of TEOS in sequence, stirring for 1.5h, finally washing with water and ethanol for several times in a crossing manner, and drying at 60 ℃ for 6h to obtain silicon dioxide coated magnetic nanoparticles Fe3O4@ SiO2;

stp15, fe obtained above ₃ O ₄ @SiO ₂ Dispersing into 100mL of anhydrous ethanol containing 5mL of APTES, and refluxing at 85 ℃ for 12h while mechanically stirring the mixture; finally, collecting the magnet, washing the magnet for a plurality of times by using water and ethanol, and drying the magnet in vacuum to obtain an amino modified magnetic material, wherein a product is marked as AMNPs;

stp02, boric acid modified Fe ₃ O ₄ @SiO ₂ The preparation of (1):

dissolving 100mg of AMNPs in 50mL of methanol, adding 200mg of FPBA and 250mg of NaBH3CN, carrying out ultrasonic dispersion, stirring for 24h at room temperature, carrying out cross washing on a product for several times by using water and methanol, and carrying out vacuum drying overnight at 60 ℃, wherein the obtained product is called FPBA-AMNPs;

stp03, dissolving lectin (ConA) in PBS with the concentration of 0.1M and the pH of 7.4 to prepare a stock solution with the concentration of 5mg/100 mL; adding 100mg of FPBA-AMNPs into the stock solution, mechanically stirring at room temperature for 15min, and storing at 4 deg.C.

The preparation of the three SERS probes comprises the following steps:

first, 100mL 0.01% of HAuCl is taken ₄ ·4H ₂ Placing O (aq) in a three-neck flask, placing in a reflux device, and heating to boil under magnetic stirring; then, 1mL of 3% trisodium citrate solution is rapidly added, and the mixture is continuously stirred for 30min, wherein the solution is wine red; finally, the obtained gold nanoparticles (AuNPs) are naturally cooled to room temperature and are filled in a clean vessel,stored at 4 ℃ before use.

The preparation of the Au-4MPBA @ Ag-P1 SERS probe also comprises the steps of adding 1mL of 1mM 4-MPBA into 10mL of the prepared AuNPs; reacting for 15min under magnetic stirring, centrifuging, removing supernatant, and dissolving with deionized water to obtain Au-4 MPBA; then adding 1mL20mM AgNO3 and 1mL0.02g/mL ascorbic acid in sequence; and after the reaction is carried out for 10min by magnetic stirring, removing unreacted excessive materials by centrifugation, dissolving the excessive materials in deionized water again to obtain Au-4MPBA @ AgNPS, and oscillating and incubating the Au-4MPBA @ AgNPS and the aptamer P1 for 10min to obtain the aptamer functionalized Au-4MPBA @ Ag-P1NPsSERS probe.

The preparation of Au @ DTNB @ Ag-P2SERS probe also included adding 1mL of 10mM DTNB to 10mL of the prepared AuNPs; magnetically stirring for reaction for 15min, centrifuging, discarding the supernatant, and dissolving again with deionized water to obtain Au-DTNB; then sequentially adding 1mL of 20mM AgNO3 and 1mL0.02g/mL ascorbic acid; and after the reaction is carried out for 10min by magnetic stirring, removing unreacted excessive materials by centrifugation, re-dissolving the excessive materials in deionized water to obtain Au-DTNB @ AgNPS, and carrying out co-oscillation incubation with the aptamer P2 for 10min to obtain the aptamer functionalized Au-4 DTNB @ Ag-P2NPsSERS probe.

The preparation of the Au @ Ag @ PB @ Ag-P3 SERS probe further comprises the step of adding 1mL0.1g/mL of ascorbic acid into 10mL of prepared AuNPs; after the reaction is carried out for 15min by magnetic stirring, 1mL of 20mM AgNO3 is added to continue the magnetic stirring for 15min, and Au @ Ag nano particles are obtained; then 1mL of 1mM FeCl3.6H2O and 1mL of 1mM K are added in this order ₄ [Fe(CN) ₆ ]·3H ₂ And O, after the reaction is carried out for 10min by magnetic stirring, centrifuging to remove the supernatant, adding 200 mu L of 10mM AgNO3, carrying out magnetic stirring for 10min, centrifuging to remove unreacted excessive materials, dissolving in deionized water again to obtain Au @ Ag @ PB @ AgNPs, and oscillating and incubating with the aptamer P3 for 10min jointly to obtain the aptamer functionalized Au @ Ag @ PB @ Ag-P3NPsSERS probe.

Au @ Ag @ PB @ Ag-P3 SERS probe has Raman signal in biological silence area, and the SERS probe is at 2081cm ^-1 Shows a strong and sharp single vibration peak which is completely separated from the complex multi-band Raman signal generated by the main endogenous biological molecules;

therefore, the characteristic peak between Au-4MPBA @ Ag-P1 SERS probe, au @ DTNB @ Ag-P2SERS probe and Au @ Ag @ PB @ Ag-P3 SERS probe three does not have mutual interference, and when adopting the above-mentioned nanoparticle of aptamer functionalization to obtain the SERS probe that can specificity discernment pathogenic bacteria, just can be used for the pathogenic bacteria of mark and three kinds of different grade types of quantitative determination. The gold-core silver-shell bimetallic nanoparticles have the uniformity and stability of Au and the strong SERS activity of Ag, and the Raman reporter molecules are wrapped between the bimetallic particles, so that a 'hot spot' structure with better signal enhancement can be generated in the gap of the core shell, and therefore, three different SERS probes selected have the characteristics of strong Raman signal, high stability and good repeatability.

As can be seen from fig. 1, fig. 1 shows that the SERS sensor for detecting pathogenic bacteria is constructed based on the combination of magnetic nanoparticles modified by ConA as a universal bacterial capture probe and three different metal nanoparticles modified by aptamers capable of specifically recognizing pathogenic bacteria as recognition probes to form a sandwich structure. The pathogenic bacteria detected by SERS depend on ConA functionalized magnetic nanoparticles as a capture probe and aptamer modified metal nanoparticles as specific recognition probes, the function of the lectin is similar to that of a capture antibody, but the lectin is cheaper than the antibody and has broad-spectrum capture capacity for bacteria, and firstly, the pathogenic bacteria in a sample are captured and separated by the ConA modified magnetic nanoparticles under the action of an external magnetic field. Then, three SERS probes of Au-MPBA @ Ag-P1, au-DTNB @ Ag-P2 and Au @ Ag-PB @ Ag-P3 modified by the aptamer are respectively added and incubated to form a sandwich structure. And finally, a sandwich structure is collected through magnetic separation, and a portable Raman spectrometer is used for detecting three different types of pathogenic bacteria, so that the capture efficiency can be greatly improved, and the specificity and the detection sensitivity are considered simultaneously.

The Au @ Ag @ PB @ AgNPs provided by the invention has very strong Raman signals in a 'biological silence zone', and three SERS probes which are mutually non-interfering are used, so that the purpose of simultaneously detecting three different types of pathogenic bacteria in a complex sample is realized, and the minimum detection limit can reach 1CFU/mL.

As shown in fig. 2, agNPs with higher SERS activity have non-uniform particle size, resulting in unstable SERS enhancement, while AuNPs with uniform distribution have lower SERS activity. The Au @ Ag core-shell nano-particle with two metals has high and stable SERS activity, and a 'hot spot' structure with stronger local plasma resonance effect is formed under the synergistic action of bimetal because Raman reporter molecules are mixed in gaps of the core shell. Therefore, three SERS probes of Au-MPBA @ Ag-P1, au-DTNB @ Ag-P2 and Au @ Ag-PB @ Ag-P3 are prepared.

As shown in FIG. 3, the design and manufacture of Au-MPBA @ Ag and Au-DTNB @ Ag SERS probes are mainly to attach the Raman reporter MPBA/DTNB on the surface of AuNPs, and then cover a silver shell on the surface. First, the nanoparticles were characterized by projection electron microscopy (TEM). Figure 3A shows that AuNPs are well distributed, with dimensions of about 19 nm (figure 3D). FIG. 3B is a TEM of SERS probe with a pronounced core-shell structure, relatively uniform size, and an average size of about 33 nm (FIG. 3E). U-Vis spectra of different nanoparticles, as shown in FIG. 3C, show red-shifts of Au-MPBA (525 nm) and AuNPs (520 nm); this change was caused by the conjugation effect of the introduced MPBA, indicating that the synthesis of Au-MPBA was successful. When the AgNPs were reduced to silver shells, the absorption peak of the AuNPs disappeared, and the characteristic absorption peak of silver appeared at 400nm, indicating that the surface of the gold nano-meter was covered with silver. In addition, SERS signals of different nanoparticle-connected 4-MPBA are compared (fig. 3F), which shows that embedding a Raman reporter molecule between gold-core silver shells can generate stronger Raman signals, and an excellent SERS probe is provided for subsequent pathogen detection.

As shown in FIG. 4, firstly we prepared Au @ Ag as the inner core of the SERS probe, and then wrapped FeCl on the outer layer ₃ ·6H ₂ O and K ₄ [Fe(CN) ₆ ]·3H ₂ And finally, in order to enable Au @ Ag-PB to be easily connected with the aptamer through Ag-S, the PB shell is wrapped by the PB shell. The TEM image of fig. 4A shows good distribution of AuNPs, approximately 32nm in size (fig. 4D). FIG. 4B is a TEM image of Au @ AgNPs with a pronounced core-shell structure, relatively uniform in size, with an average size of about 48nm (FIG. 4E). FIG. 4C is TEM image of Au @ Ag-PB NPs, it can be clearly seen that a thin PB shell is wrapped on the Au @ Ag shell,about 2nm thick, au @ Ag-PB NPs average size about 52nm (FIG. 4F), FIG. 4G is the TEM image of Au @ Ag-PB @ Ag NPs, the size of the nanoparticles is 69nm (FIG. 4I), 17nm more than Au @ Ag-PB NPs, indicating successful Ag shell wrapping at the outer layer of Au @ Ag-PBNPs.

Design and preparation of magnetic nanoparticles mainly comprising Fe ₃ O ₄ @SiO ₂ The synthesis of (2) is carried out, the linking of amino groups, the modification of boronic acid and the functionalization of ConA are carried out. For the measurement of morphology, fe was measured by TEM and Scanning Electron Microscope (SEM) ₃ O ₄ ，Fe ₃ O ₄ @SiO ₂ FPBA-AMNPs were characterized. As shown in FIG. 5A (a), the Solvothermal method produced Fe ₃ O ₄ The magnetic nano-particles are in a regular spherical shape, the particle size is uniform, the dispersity is good, and the average particle size is 268nm. FIG. 5A (b) shows Fe ₃ O ₄ The surface of the nano-particles is coated with a layer of SiO with uniform thickness ₂ The thickness of the layer and the shell layer is about 28nm ₂ Not only increases the particle size of the magnetic nanoparticles, but also increases the hydrophilicity, dispersibility and stability, and it can be seen from the SEM in FIG. 5 (c) that Fe is present ₃ O ₄ Uneven surface coated with SiO ₂ The rear surface is smooth fig. 5 (d). Fourier transform Infrared Spectroscopy (FT-IR) for studying Fe ₃ O ₄ 、Fe ₃ O ₄ @SiO ₂ Amination of AMNPs and FPBA-AMNPs and formation of boric acid grafts.

FIG. 5B shows Fe ₃ O ₄ Characteristic peak of (1) is 581cm ^-1 Here, this is the stretching vibration peak of Fe-O; coated with SiO ₂ After that, at 942cm ^-1 And 1089cm ^-1 Two groups of characteristic peaks appear and are respectively attributed to the stretching vibration of Si-O-Si and Si-O-H; amino group modified at 1548cm ^-1 The band of the amino group belongs to the N-H stretching vibration of the amino group, which shows that the amino group is successfully modified in SiO ₂ Of (2) is provided. Furthermore, 1368cm ^-1 And 1520cm ^-1 The two new peaks at (a) are the stretching peak of B-O and the stretching vibration peak of C = C on the benzene ring, respectively, indicating that FPBA has been grafted onto the amino group. Fe was identified by X-ray diffraction (XRD) analysis ₃ O ₄ 、Fe ₃ O ₄ @SiO ₂ And crystalline phases of FPBA-AMNPs. As shown in FIG. 5C, XRD of the prepared materials all showed Fe ₃ O ₄ The 6 characteristic diffraction peaks at 2 θ =30.1 °, 35.4 °, 43.2 °, 53.4 °, 57.1 °, and 62.5 ° may be indicated as (220), (311), (400), (422), (511), and (440), respectively (JCPDS card: 19-0629). Indicating that the crystal structure of magnetite remains essentially unchanged during the preparation of the material. In addition, with Fe ₃ O ₄ In contrast, despite Fe ₃ O ₄ @SiO ₂ And FPBA-AMNPs, and the diffraction peak position of the FPBA-AMNPs is not changed, and no new diffraction peak is generated, but the peak intensity is gradually reduced, so that the surface of the FPBA-AMNPs is coated with an amorphous material. To further determine the FPBA vs Fe ₃ O ₄ @SiO ₂ -NH ₂ Modification of the surface, using X-ray photoelectron Spectroscopy (XPS) for Fe ₃ O ₄ And FPBA-AMNPs were subjected to elemental analysis. As can be seen from FIG. 5D, fe ₃ O ₄ The XPS plots show peaks of Fe2p and O1s at 712 and 545eV, and show peaks of Si2s and Si2p at 151 and 100eV, respectively, from the XPS plot of FPBA-AMNPs, indicating that Fe ₃ O ₄ Is coated with a layer of SiO ₂ The N1s peak at 399.1eV indicates that APTES has been immobilized on Fe ₃ O ₄ @SiO ₂ Due to the low abundance of B element, a weak B1s peak (as seen from the inset) appeared at 190.4eV, indicating that FPBA has been immobilized in Fe ₃ O ₄ @SiO ₂ A surface.

To further characterize the enrichment and isolation capabilities of our synthesized FPBA-AMNPs, fe was measured using the hysteresis loop test (VSM) ₃ O ₄ 、Fe ₃ O ₄ @SiO ₂ And magnetization curves of FPBA-AMNP, as shown in FIG. 6A; all show good magnetic properties, wherein Fe ₃ O ₄ Has a Magnetic Saturation (MS) value of 67.89emu/g; wrapping SiO ₂ After that, the MS value is obviously reduced (43.70 emu/g) due to the fact that the surface is coated with a layer of SiO ₂ A thin layer. The Ms value (43.39 emu/g) is not obviously reduced after amino modification and boric acid grafting, which indicates that the influence of chemical bonds or chemical groups on the magnetic response is not large.

To determine the binding affinity of FPBA-AMNPs to ConAForce, we evaluated the binding isotherms of ConA and FPBA-AMNPs; as shown in FIG. 6B, the maximum adsorption amount of the FPBA-AMNPs to ConA is 29.8mg/g, which indicates that the prepared FPBA-AMNPs have better adsorption capacity. The adsorption isotherms and the quantitative analysis of the binding affinity were analyzed using Scatchard plots. First, the adsorption amount Qe of FPBA-AMNPs in ConA solutions of different concentrations and the concentration of ConA at adsorption equilibrium were measured, and then the value of Qe/[ ConA ] was determined]Plotted as ordinate and Qe abscissa, and the linearly fitted equation is Scatchard's equation for equilibrium experiments, and the dissociation constant Kd is calculated from the inverse of the slope. Fig. 6C shows that Scatchard plot analysis provided a dissociation constant (Kd) of (2.34 ± 0.09) × 10 at pH =7.4 ^-7 And M. The FPBA-AMNPs prepared by the method have strong bonding strength with ConA, so that a stable adsorption material is provided for enrichment and separation of pathogenic bacteria. In order to be cost-effective. The reusability of magnetic materials is also of interest, and the boric acid affinity linker protein can achieve the effects of adsorption and desorption by adjusting the pH value. Therefore, we studied the regenerability of FPBA-AMNPs through adsorption-desorption experiments, and as can be seen from fig. 6D, after 5 regeneration cycles, SERS signals of FPBA-AMNPs adsorbing ConA are slightly decreased, about 6.76%, which indicates that after multiple adsorption-desorption cycles, the stability of MNPs is still good, indicating that the regeneration of particles does not damage the fixed structure under the condition of increasing or decreasing pH. Therefore, MNPs have good reusability, making SERS sensors less costly.

In order to prove that the characteristic Raman peaks of three different SERS probes do not interfere with each other, as shown in FIG. 7 (A), au-DTNB @ Ag, au-MPBA @ Ag and Au @ Ag-PB @ Ag are respectively at 1334cm ^-1 、1078cm ^-1 、2081cm ^-1 The strong and sharp Raman characteristic peak is located, because PB only has SERS signals with clean background and high peak intensity in a 'biological silence region', the SERS signals are completely free of interference with two Raman reporter molecules of Au-DTNB @ Ag and Au-MPBA @ Ag, and the method is the basis for realizing simultaneous triple joint detection of pathogenic bacteria. When SERS detection is carried out on mixed bacteria with different concentrations of staphylococcus aureus, escherichia coli and pseudomonas aeruginosa in the mixed SERS probe of the three Raman reporter molecules, as shown in fig. 7 (B), when the staphylococcus aureus and the large intestine rod are in the same stateThe concentration of the bacteria and the pseudomonas aeruginosa is from 1X 10 ¹ ～1×10 ⁷ CFU/mL, the Raman signal intensity of the mixed SERS is gradually increased, and a good linear relation is presented. FIG. 7 (D, E, F) the linear regression equation for Staphylococcus aureus is Y =872.44+2129.30X with a limit of detection (LOD) of 1CFU/mL; the linear regression equation for E.coli is Y =909.85+2070.66X, and the detection Limit (LOD) is 1CFU/mL; the linear regression equation of the pseudomonas aeruginosa is Y =1214.75+2634.14X, and the detection Limit (LOD) is 1CFU/mL. In addition, as shown in fig. 7C, the mixed SERS probe accurately identifies staphylococcus aureus, escherichia coli, and pseudomonas aeruginosa, and has very high raman intensity, which confirms that the mixed SERS probe has excellent specificity for staphylococcus aureus, escherichia coli, and pseudomonas aeruginosa, and has no cross reaction with other pathogens. Based on our demonstrated bioanalysis of staphylococcus aureus, escherichia coli, and pseudomonas aeruginosa, many similar bioanalytical targets can be found by using a mixed SERS probe. Therefore, the synthesized hybrid SERS probe has great potential in identifying wide components of various organisms in a complex biological sample, and has great reusability, extraordinary sensitivity and special selectivity

In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for simultaneously detecting three pathogenic bacteria based on lectin and aptamer dual recognition is characterized by comprising the following steps:

stp1, incubating the lectin magnetic nanoparticles and three different types of pathogenic bacteria for 30min, and washing by using a PBS (phosphate buffer solution) under the action of an external magnetic field to remove the unbound pathogenic bacteria;

stp2, preparing three aptamer functionalized surface enhanced SERS probes, incubating the surface enhanced SERS probes with the lectin magnetic nanoparticles captured with pathogenic bacteria for 30min to form a sandwich structure,

the three SERS probes comprise an Au-MPBA @ Ag-P1 SERS probe, an Au @ DTNB @ Ag-P2SERS probe and an Au @ Ag @ PB @ Ag-P3 SERS probe;

stp3, washing 3 times with PBS to remove unbound SERS probes, magnetic separation followed by reconstitution of the original volume, and analysis using a portable raman spectrometer.

2. The method for simultaneously detecting three pathogenic bacteria based on the dual recognition of the lectin and the aptamer according to claim 1, wherein the preparation method of the lectin magnetic nanoparticles comprises the following steps:

stp01, amino-modified magnetic nanoparticles (Fe) ₃ O ₄ @SiO ₂ -NH ₂ ) The preparation of (1):

stp11, 2.1g of ferric chloride hexahydrate (FeCl) ₃ ·6H ₂ O), 4g of anhydrous sodium acetate (NaAc) and 1g of polyethylene glycol are added into 60mL of ethylene glycol, stirred and dissolved completely, and then placed into a 100mL high-pressure reaction kettle with a polytetrafluoroethylene lining for reaction for 8h at the temperature of 195-198 ℃;

stp12, cooling, washing with water and ethanol for several times, and drying in vacuum drying oven at 60 deg.C to obtain magnetic ferroferric oxide nanoparticles (Fe) ₃ O ₄ NPs)；

Stp13, 200mgFe ₃ O ₄ NPs are added into 60mL of absolute ethyl alcohol and 15mL of water, and are fully dispersed by ultrasonic;

stp14, adding 2.8mL ammonia water and 0.4mL TEOS (ethyl silicate) in sequence, stirring for 1.5h, washing with water and ethanol for several times, and drying at 60 deg.C for 6h to obtain silicon dioxide (SiO) ₂ ) Coated magnetic nanoparticles Fe ₃ O ₄ @SiO ₂ NPs；

Stp15 and Fe obtained above ₃ O ₄ @SiO ₂ Dispersing into 100mL of anhydrous ethanol containing 5mL of Aminopropyltriethoxysilane (APTES), and refluxing at 60 deg.C for 12h while mechanically stirring the mixture; finally collecting the magnetic iron, washing the magnetic iron with water and ethanol for a plurality of times, and drying the magnetic iron in vacuum to obtain the amino modified magnetic material, wherein the product is marked as (Fe) ₃ O ₄ @SiO ₂ -NH ₂ )AMNPs；

Stp02 and preparation of boric acid modified AMNPs:

100mg of AMNPs are dissolved in 50mL of methanol, and 200mg of formylphenylboronic acid (FPBA) and 250mg of sodium cyanoborohydride (NaBH) ₃ CN), stirring for 24 hours at room temperature after ultrasonic dispersion, alternately washing the product for a plurality of times by using water and methanol, and drying overnight in vacuum at 60 ℃ to obtain a product called FPBA-AMNPs;

preparation of Stp03, lectin functionalized FPBA-AMNPs

Dissolving lectin (ConA) in PBS (0.1M, pH 7.4) to obtain stock solution with concentration of 5mg/100 mL; adding 50mg FPBA-AMNPs into the stock solution, mechanically stirring at room temperature for 15min to obtain ConA @ FPBA-AMNPs, and storing at 4 deg.C.

3. The method for simultaneously detecting three pathogenic bacteria based on the dual recognition of lectin and aptamer according to claim 1, wherein the method comprises the following steps:

the preparation of the three SERS probes comprises the following steps:

first, 100mL of 0.01% chloroauric acid (HAuCl) was taken ₄ ·4H ₂ O) placing the mixture in a three-neck flask, placing the mixture in a reflux device, and heating the mixture to boiling by magnetic stirring; then add 1mL of 3% lemon quicklyStirring the trisodium acid solution for 30min, wherein the solution is wine red; finally, the obtained gold nanoparticles (AuNPs) are naturally cooled to room temperature, filled in a clean vessel and stored at 4 ℃ before use.

4. The method for simultaneously detecting three pathogenic bacteria based on lectin and aptamer dual recognition is characterized in that the preparation of the Au-4MPBA @ Ag-P1 SERS probe further comprises the step of adding 1mL of 1mM 4-MPBA into 10mL of prepared AuNPs; magnetically stirring for reaction for 15min, centrifuging, removing supernatant, and dissolving again with deionized water to obtain Au-4 MPBA; then 1mL of 20mM AgNO3 and 1mL of 0.02g/mL ascorbic acid are added in sequence; after the reaction is carried out for 10min by magnetic stirring, the unreacted excessive materials are removed by centrifugation and are dissolved in deionized water again to obtain Au-4MPBA @ AgNPs, and the Au-4MPBA @ AgNPs and the aptamer P1 are vibrated and incubated for 10min together to obtain the aptamer functionalized Au-4MPBA @ Ag-P1NPsSERS probe.

5. The method for simultaneously detecting three pathogenic bacteria based on double recognition of lectin and aptamer according to claim 3, wherein the preparation of the Au @ DTNB @ Ag-P2SERS probe further comprises adding 1mL of 10mM DTNB into 10mL of prepared AuNPs; magnetically stirring for reaction for 15min, centrifuging, removing supernatant, and dissolving again with deionized water to obtain Au-DTNB; then 1mL of 20mM silver nitrate (AgNO) was added sequentially ₃ ) And 1mL0.02g/mL of ascorbic acid; and after the reaction is carried out for 10min by magnetic stirring, removing unreacted excessive materials by centrifugation, re-dissolving the excessive materials in deionized water to obtain Au-DTNB @ AgNPs, and carrying out co-oscillation incubation with the aptamer P2 for 10min to obtain the aptamer functionalized Au-4 DTNB @ Ag-P2NPsSERS probe.

6. The method for simultaneously detecting three pathogenic bacteria based on lectin and aptamer double recognition is characterized in that the preparation of the Au @ Ag @ PB @ Ag-P3 SERS probe further comprises the step of adding 1mL0.1g/mL of ascorbic acid into 10mL of prepared AuNPs; after magnetically stirring the reaction mixture for 15min, 1mL of 20mM AgNO was added ₃ Continuing magnetic stirring for 15min to obtain Au @ Ag nanoparticles;

1mL of 1mM ferric chloride hexahydrate (FeCl) was then added in sequence ₃ ·6H ₂ O) and 1mL of 1mM potassium ferrocyanide (K) ₄ [Fe(CN) ₆ ]·3H ₂ O), magnetically stirring for 10min, centrifuging to remove supernatant, adding 200 μ L of 10mM AgNO ₃ Magnetic stirring 10min, the centrifugation is got rid of unreacted excessive material and is redissolved in order to obtain Au @ Ag @ PB @ AgNPs in the deionized water, vibrates jointly with aptamer P3 and incubates 10min, obtains the aptamer functionalized Au @ Ag @ PB @ Ag-P3NPsSERS probe.

7. The method for simultaneously detecting three pathogenic bacteria based on dual recognition of lectin and aptamer according to claim 1, wherein the concentration of PBS in Stp1 and Stp3 is 0.1M, and the pH =7.4;

the preparation of the 0.1M PBS solution with pH =7.4 was specifically: 7.8g of sodium dihydrogen phosphate (NaH) was accurately weighed ₂ PO ₄ ) 475mL of deionized water is added, stirred and dissolved sufficiently, then 2.0g of NaOH is weighed to prepare 1.0M NaOH solution, the pH value of the solution is adjusted to 7.4, the solution is transferred to a 500mL volumetric flask, deionized water is added to 500mL, and the solution is mixed sufficiently for standby.

8. The method for simultaneously detecting three pathogenic bacteria based on the dual recognition of lectin and aptamer according to claim 1, wherein in Stp3, a Raman spectrum is recorded with a 785nm laser with 200mW power, and the integration time is 500ms and 3 times of accumulation.

9. A method for simultaneously detecting three pathogenic bacteria based on lectin and aptamer dual recognition, as claimed in claim 8, wherein the Raman spectra recorded are averaged over 5 replicates and all Raman spectra are baseline corrected.