CN105891159A - Molecularly imprinted-SPR sensing method based on histamine rapid detection - Google Patents

Abstract

The invention relates to the field of SPR sensor chips, in particular to a histamine molecularly imprinted SPR sensor chip and a preparation method thereof. According to the histamine molecularly imprinted SPR sensor chip, a method for spin coating of a grafting initiator is adopted, and a histamine MIP membrane is prepared on the surface of an SPR chip in an in-situ polymerization mode. The histamine molecularly imprinted SPR chip has high-specificity adsorptivity on histamine and has obvious response signal changes for low-concentration histamine in a sample. The prepared histamine molecularly imprinted SPR chip is adopted for analyzing histamine residues, the steps are simple, the analysis time is short, and high specificity and sensitivity are achieved to analysis at low concentration.

Description

Molecular engram-SPR sensorgram the method quickly detected for histamine

Technical field

The present invention relates to a kind of spr sensor detection method, belong to field of biosensors.It is specifically related to quickly detect histamine molecular engram SPR Sensor chip and method for sensing.

Background technology

Histamine is a kind of spontaneous material, can produce in many veterinary antibiotics, medicated beer, fish, cheese, fermented food and red wine Histamine (Lehane&Olley, 2000), in these food, the concentration of histamine is relatively low, but after rotting, the substantial amounts of output histamine of meeting, The concentration of histamine can exceed the 50mg/100g of toxic level, causes alimentary toxicosis (Lehane et al., 2000) after eating.Group in the freshest fish After amine content is the lowest, but fish rots, also can produce substantial amounts of histamine (Fernandez-Salguero&Mackie, 1987；Frank, Yoshinaga, &Nip, 1981), therefore, histamine is also proposed as chemical index (L ó pez-Sabater, Rodr í guez, Roig-Sagu the é s, & of Estimation of The Fish Freshness Mora-Ventura, 1994).The standard of the edible fishes histamine that food and drug administration sets up is 5mg/100g, and Histamine concentrations exceedes this mark Standard is prohibited to sell (Food and Drug Administration, 1996).Therefore, the detection of histamine is extremely important to food industry and food safety. In order to provide scientific basis to HACCP (HACCP) certification during food processing and production, at food and food-processing industry, soon Speed, detection histamine accurate, reliable seem the most necessary.

A lot of methods is had to be developed for detecting histamine.The technology detecting histamine the most frequently used has: high performance liquid chromatography (HPLC) (Yoshitake Et al., 2003), gas chromatogram (GC) (Keyzer, Wolthers, Muskiet, Breukelman, Kauffman ， &Vries, 1984), enzyme connection Immunosorbent adsorption test (ELISA) (Ujike et al., 1999) etc., these method agents useful for same, vessel are more, apparatus expensive, complex operation, Time-consuming, sensitivity is low.Such as, although ELISA method provides high sample size analysis, loaded down with trivial details and time-consuming operating procedure is the shortcoming that it is main, Another shortcoming of the method is that it needs a long incubation time, owing to, on elisa plate, immunoreation slowly causes incubation time longer.One Plant based on the instrument producing plasma resonance (SPR) phenomenon on thin golden film surface, have been used for detecting unmarked histamine in real time.SPR is Be proved to be the most promising method of one, may be used for substituting some traditional methods (Homola, 2008；Shankaran&Miura, 2007). SPR has become as a conventional tool (Cheng, Huang ， &Duan, 2003) of life sciences and drug research laboratory.Furthermore, it is possible to make The binding specificity between chemical substance and kinetics and the quantitative determination (Myszka, 1997) to chemical substance is measured with spr sensor.As Really the chip of spr sensor is modified by target molecule specific receptor, and spr sensor can also be by the biosensor as high selectivity and susceptiveness. Such as, Yan Li et al. application based on indirect competition immunoreactive surface plasmon resonance biosensor detection histamine (Li, Kobayashi, Furui, Soh, Nakano ， &Imato, 2006), but this sensor utilizes biological identification element, and such as antibody or enzyme, but this sensor-based system limits its application (Kandimalla&Ju, 2004；Yano&Karube, 1999).First, biological identification element is expensive, it is difficult, due to storage etc. to prepare Reason may cause target molecule the most effective.Additionally, biological identification element requires height to solvent, temperature and acid-base value, can be with solvent, temperature The change of degree and pH value becomes unstable, and the recognition component of synthetic then has good stability (Owens, Karlsson, Lutz, & Andersson, 1999).The method of the standard test histamine that China is existing is GB/T5009.45-2003 and GB " five kinds of biogenic amine such as water quality histamine Mensuration high performance liquid chromatography " (GB/T21970-2008), there is presently no the standard of histamine in application spr sensor detection food, at home In outer literature search, in application spr sensor detection food, the method for histamine is also little, and spr sensor has easy and simple to handle, highly sensitive, fast The advantages such as speed, the spr sensor detection method hence setting up histamine in food is the most necessary.

Summary of the invention

It is an object of the invention to provide a kind of histamine molecular engram spr sensor detection method.

Technical scheme is summarized as follows: a kind of histamine molecular engram spr sensor chip is by histamine (Histamine): monomer (MAA): The method that cross-linking agent (EGDMA) uses spin coating graft initiator with 1: 2: 4 ratio, prepares the MIP film of histamine at SPR chip surface in-situ polymerization. Respectively with MIP film, SPR chip film modified for NIP as the recognition component of histamine, prepare variable concentrations (25,50,100,250,500,1000 Ng/mL) histamine PBS solution, detects with spr sensor, records the data obtained, each horizontal survey 3 times, obtains variable concentrations histamine The SPR produced responds signal, draws out response curve, Criterion curve, then evaluates each film absorption property to histamine.Use this sensor It is: 25ng/mL～1000ng/mL that detection limit value is: 25ng/mL to the detection range of histamine.

Advantages of the present invention: first, the synthesis cost of MIPs is the lowest, well below the preparation of antibody；Second, MIPs have preferable stability, Can apply in extreme temperature and pH environment；3rd, MIPs are prepared by the method by non-covalent bond, have good reproducibility.Should Sensor not only has stronger specific recognition and binding ability to histamine molecule, and has good repeatability and long-time stability, its energy Enough it is repeated several times and histamine is detected.

Accompanying drawing explanation

The experiment flow figure of Fig. 1 spr sensor based on MIP film detection histamine

Fig. 2 histamine and analog thereof

Fig. 3 scanning electron microscope (SEM) on SPR chip modify NIP film (A) and the phenogram of MIP film (B)

The histamine structural representation of Fig. 4 difference protonation

The impact (note: the concentration of histamine is 100ng/mL) on spr sensor based on MIP film detection histamine of Fig. 5 pH value

The absorption response curve of Fig. 6 MIP-SPR sensor detection variable concentrations histamine

The response curve of Fig. 7 MIP-SPR sensor detection variable concentrations histamine and standard curve (n=3)

The absorption response curve of Fig. 8 MIP-SPR sensor continuous detecting 500ng/mL histamine

The selectivity ratios that histamine is detected by Fig. 9 MIP film and NIP film is relatively (n=3)

The comparison of the SPR angle change of Figure 10 histamine and analog

Detailed description of the invention

Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, It is conventional method.Reagent used in following embodiment and material, if no special instructions, all can be commercially available from conventional reagent company.

Embodiment 1

The preparation of MIP film

After SPR chip is cleaned up, put into immersion 1h in the Piranha solution that 5mL newly prepares, more carefully rush with distilled water, dehydrated alcohol Washing, nitrogen is standby after drying up.

The method using spin coating graft initiator, prepares the MIP film of histamine at SPR chip surface in-situ polymerization, and manufacturing process is as shown in Figure 1.

(1) preparation of prepolymerization liquid: take the DMSO (40 × 25mm) in weighing botle of 5.0mL, add template molecule histamine (0.2mmol), Monomer MAA (0.4mmol), cross-linking agent EGDMA (0.8mmol), after ultrasonic deoxygenation 10min, then use N₂Deoxygenation 10min.

(2) 30mg initiator (AIBN) and 3mg adhesive agent (PVC) are dissolved in 3mL oxolane (THF) solution.Take out 10 0 μ L drips in SPR chip surface center lentamente, and then on SC-1B type glue evenning table, spin coating is uniform, and spin coating rotating speed is 58.83 × 21. 60rpm, spin-coating time 60s.The ratio respectively formed during the synthesis of MIP film is histamine (Histamine): monomer (MAA): cross-linking agent (EG DMA) ratio of synthesis MIP film is 1: 2: 4.

(3) by spin coating, the SPR chip of spin coating liquid immerses rapidly in prepolymerization liquid, continues logical N2 deoxygenation 10min, deadend weighing botle, It is placed in electric heating constant-temperature blowing drying box, 60 DEG C of reaction 16h.After polyreaction, SPR chip is immersed in methanol: glacial acetic acid=9: 1 (v/v) Mixed solution in, eluted template molecule, each 40min, eluting 4 times, then again with methanol eluant solution 2 times, finally with dehydrated alcohol, ddH₂O Rinse rear N well₂Dry up standby.It is added without histamine, by the NIP film that same method preparation is corresponding.

The surface topography of SPR chip surface polymeric film is characterized by Quanta 200FEG Flied emission environmental scanning electron microscope (SEM) As shown in Figure 3.

Embodiment 2

SPR detects

The specified conditions that surface plasma body resonant vibration occurs are that thin metal (gold or silver) film is put into laser beam, when incident illumination is monochromatic light and p- Polarized light when injecting metallic film surface at an angle, generation is vibrated and produces resonance absorption energy by the free electron of metal surface, this draws The incident angle of light playing surface plasma body resonant vibration generation is referred to as SPR angle, detects surface plasma body resonant vibration by the intensity measuring reflection light and occurs Effect, when incident illumination is injected with SPR angle, the intensity of reflection light can decline rapidly and reaches minimum, and the size at SPR angle depends on sensing chip table The refractive index in face.This surface plasma resonance sensor, based on Kretschmann structure, can be measured binding events in real time, measures During without labelling.Polarized light is to be launched by the laser diode of 3mW to produce, and wavelength is 670nm, and detection range is 4000 milli degree (mo), Having the flow cell (channel 1 and channel 2) of two same volumes on SPR chip, two flow cells are each independent, can add different Sample, after polarized light is injected and scribbled on the SPR chip of gold film, the intensity of reflection light can change, by the measurement reflection light SPR angle time the most weak Realize the detection to sample, test all of sampling and all completed by Autosampler, be divided into 4 stages: baseline (baseline), In conjunction with (association), dissociate (dissocication), regenerates (regeneration).By being 25ng/mL-1000ng/mL to concentration Histamine solution detect, we delineate to histamine detection dose-effect curve and standard curve, after concentration is carried out logarithmic transformation, paint Having made standard curve, formula is: y=29.00lgx-31.90, R²=0.997, the detection range of histamine is: 25ng/mL-1000ng/mL, Detection limit value (LOD): 25ng/mL.Such as accompanying drawing 7.

The pH value impact on MIP film identification histamine, the figure shows (pH 1-14) the hydrogen bond shape between histamine and MIP film in wide pH value range The probability become, when pH value is positioned at 7-9, MIP film is best to the adsorption effect of histamine, such as accompanying drawing 5, responds the strongest when pH is 8.

We verify the performance of MIP-SPR sensor by the histamine of detection variable concentrations.During measurement, in MIP-SPR sensor distribution channel it is It is filled with PBS, after baseline stability, variable concentrations histamine is detected the most respectively.The absorption response of MIP-SPR sensor detection variable concentrations histamine As shown in Figure 6, SPR response curve changes along with the increase of Histamine concentrations curve in gradient as seen from the figure.

MIP film completes regeneration by 0.1M HCl regenerated liquid after having detected histamine, thus reaches to remove target molecule and obtain the purpose again detected. 0.1M HCl is well suited as the regenerated liquid of this sensor, and it can be good at dissociating the hydrogen bond that histamine is combined with MIP film thus the purpose reaching regeneration. After adding histamine solution, after MIP film is to the absorption of histamine and removing non-specific adsorption, SPR response signal reaches steady statue, now adds After entering regenerated liquid, spr signal can decline rapidly, more repeatedly rinse through PBS and be eventually returned to baseline.Application MIP-SPR sensor continuous detecting 500 Ng/mL histamine, reaction result such as accompanying drawing 8, MIP film detection histamine has good stability and reproducibility.

Respectively with MIP film, SPR chip film modified for NIP as recognition component, the histamine solution of detection variable concentrations, each measurement of concetration 3 times, Comparative result as shown in Figure 9, as seen from the figure, MIP film to the selectivity of histamine significantly better than NIP film, MIP-SPR sensor to histamine Sensitivity is about 5 times of NIP-SPR sensor, it may be said that the MIP film of bright making is good to histamine adsorption.

It is the 5-hydroxy tryptamine of 10 μ g/mL, 1-imidazoleacetic acid, 5-HIAA, histidine solution by the concentration of preparation, with MIP film, MIP Film modified spr sensor detects respectively, each horizontal detection 3 times, and testing result drawing such as accompanying drawing 10, the concentration of histamine analog is 10 times of histamine solution, testing result shows: the MIP film of preparation is good to the selectivity of histamine, and detection histamine is had good specificity.

In order to study the practicality of this MIP-SPR sensor, we carry out recovery testu using fish as actual sample, it is thus achieved that good recovery Rate, the response rate is between 89.33%-105.33%, and relative standard deviation (RSD) is between 3.84%-4.81%.

Table 2-2 carries out recovery testu (n=3) using fish as actual sample

Claims (4)

1. one kind uses the method that spin coating graft initiator prepares histamine MIP film at SPR chip surface, it is characterised in that its processing procedure is by following steps Composition:

(1) preparation of prepolymerization liquid: take the DMSO (40 × 25mm) in weighing botle of 5.0mL, add template molecule histamine (0.2mmol), Monomer MAA (0.4mmol), cross-linking agent EGDMA (0.8mmol), after ultrasonic deoxygenation 10min, then use N₂Deoxygenation 10min.

(2) 30mg initiator (AIBN) and 3mg adhesive agent (PVC) are dissolved in 3mL oxolane (THF) solution.Take out 100 μ L Dripping in SPR chip surface center lentamente, then on SC-1B type glue evenning table, spin coating is uniform, and spin coating rotating speed is 58.83 × 21.60rpm, Spin-coating time 60s.

(3) by spin coating, the SPR chip of spin coating liquid immerses rapidly in prepolymerization liquid, continues logical N₂Deoxygenation 10min, deadend weighing botle, it is placed in In electric heating constant-temperature blowing drying box, 60 DEG C of reaction 16h.After polyreaction, SPR chip is immersed in methanol: glacial acetic acid=9: 1 (v/v's) is mixed Closing in solution, eluted template molecule, each 40min, eluting 4 times, then again with methanol eluant solution 2 times, finally with dehydrated alcohol, ddH₂O Rinse rear N well₂Dry up standby.It is added without histamine, by the NIP film that same method preparation is corresponding.

2. the method preparing histamine MIP film, is characterized in that the ratio that MIP film respectively forms when synthesizing is histamine (Histamine): monomer (MA A): the ratio of cross-linking agent (EGDMA) synthesis MIP film is 1: 2: 4.

3. molecular engram-SPR sensorgram method that histamine quickly detects, is characterized in that by the histamine that concentration is 25ng/mL～1000ng/mL Solution detects, and draws out standard curve, and formula is: y=29.001gx-31.90, R²=0.997, the detection range of histamine is: 25ng/mL～ 1000ng/mL, detection limit value (LOD): 25ng/mL.

4. molecular engram-SPR sensorgram method that a histamine quickly detects, it is characterised in that the optimal pH of detection histamine solution is 8.