CN115452802A - Preparation method of SERS sensor based on liquid drop hydrogel microspheres and application of SERS sensor in blood glucose detection - Google Patents

Abstract

The invention relates to the technical field of biosensors, in particular to a preparation method of a SERS sensor based on liquid drop hydrogel microspheres and application of the SERS sensor in blood glucose detection. Silver nanoparticles (AgNPs) for modifying Glucose Oxidase (GOX) are used as SERS nano probes to be loaded in liquid drop hydrogel microspheres to obtain an SERS sensor; the principle that the Raman enhancement effect of AgNPs is weakened due to the fact that the AgNPs are etched by gluconic acid (a reaction product of glucose and GOX) is utilized, and rapid and direct detection of the glucose is achieved. The invention has good specificity and sensitivity, the detection limit is 0.1mM, and the linear range is 0-20mM. The hydrogel matrix can protect the silver nanoparticles from being polluted by the surrounding environment and enhance the chemical stability of the silver nanoparticles serving as an SERS substrate; the silver nanoparticles are uniformly distributed in the hydrogel microspheres, so that the repeatability of SERS detection is greatly improved. The sensing method consumes a small amount of samples, has quick response time, and has good practical application prospect in the field of clinical liquid biopsy.

Description

Translated fromChinese

一种基于液滴水凝胶微球的SERS传感器的制备方法及其在血糖检测中的应用Preparation method of a SERS sensor based on droplet hydrogel microspheres and its application in bloodApplication in Sugar Detection

技术领域technical field

本发明涉及生物传感器技术领域，尤其涉及一种基于液滴水凝胶微球的SERS传感器的制备方法及其在血糖检测中的应用。The invention relates to the technical field of biosensors, in particular to a method for preparing a SERS sensor based on droplet hydrogel microspheres and its application in blood sugar detection.

背景技术Background technique

表面增强拉曼散射(SERS)作为一种无创、指纹性光谱，由于其具有非常高的灵敏度和选择性，近年来被广泛应用于生物分析。表面拉曼散射是指金属纳米结构通过表面等离子体共振(SPR)增强表面电磁场，从而使表面附近分子的拉曼散射强度显著提高的一种现象。表面增强拉曼散射光谱用于检测生物小分子包括两大类方法：直接法和间接法。直接法是通过直接测定目标小分子的本征SERS信号获得对应的目标物浓度；间接法往往需要设计对待测物有识别响应的纳米探针，通过采集探针分子的SERS信号间接反映待测物的浓度。SERS直接检测的主要问题是光谱繁杂，从复杂光谱中提取有用的信息极具挑战性。其次，当SERS基底置于空气或复杂生物样本中时，环境中的污染物很容易吸附在SERS基底上，产生背景信号，干扰目标物的测定。另一个需要解决的关键问题是如何提高SERS检测的重现性。SERS检测重复性差通常是由于溶液中金属纳米粒子的聚集程度不同及目标分子在纳米粒子上的不均匀分散。上述问题也是SERS检测生物小分子面临的重要挑战。Surface-enhanced Raman scattering (SERS), as a noninvasive, fingerprinting spectroscopy, has been widely used in biological analysis in recent years due to its very high sensitivity and selectivity. Surface Raman scattering refers to a phenomenon in which metal nanostructures enhance the surface electromagnetic field through surface plasmon resonance (SPR), thereby significantly increasing the Raman scattering intensity of molecules near the surface. Surface-enhanced Raman scattering spectroscopy for the detection of small biomolecules includes two broad categories of methods: direct and indirect. The direct method is to obtain the corresponding target concentration by directly measuring the intrinsic SERS signal of the target small molecule; the indirect method often needs to design a nanoprobe that has a recognition response to the analyte, and indirectly reflects the analyte by collecting the SERS signal of the probe molecule. concentration. The main problem of SERS direct detection is that the spectrum is complicated, and it is extremely challenging to extract useful information from complex spectra. Secondly, when the SERS substrate is placed in the air or complex biological samples, the pollutants in the environment are easily adsorbed on the SERS substrate, resulting in background signals and interfering with the determination of the target. Another key issue that needs to be addressed is how to improve the reproducibility of SERS detection. The poor repeatability of SERS detection is usually due to the different aggregation degrees of metal nanoparticles in solution and the uneven dispersion of target molecules on nanoparticles. The above problems are also important challenges for the detection of small biological molecules by SERS.

微流控技术和SERS的结合很好地解决了上述问题，在微流控芯片通道中便于控制金属纳米粒子的聚集及待测分子在SERS基底上的均匀吸附。近年来，就复杂样品的高选择性和高灵敏检测而言，以微流控液滴为模板制备的水凝胶微球已得到迅速的发展。微流控技术能够以高通量的方式产生均匀的单分散液滴，这些液滴被用作模板，以制备具有各种物理和化学特性的聚合物微球。通过将单体与光引发剂共同装载在液滴中，采用紫外光照射使其在液滴中进行交联聚合，在很短的时间内就能获得大量水凝胶微球。产生的微球具有均匀的尺寸，可调的孔径，良好的单分散性和高产量。通过改变液滴中单体与水的质量比，能够制备出具有不同孔径的水凝胶微球，可用于体液中生物小分子或金属离子的选择性检测，实现了直接检测生物体液的目标，避免了复杂的样品前处理步骤。The combination of microfluidic technology and SERS solves the above problems well, and it is convenient to control the aggregation of metal nanoparticles and the uniform adsorption of the molecules to be tested on the SERS substrate in the channel of the microfluidic chip. In recent years, hydrogel microspheres prepared with microfluidic droplets as templates have been developed rapidly for high selectivity and high sensitivity detection of complex samples. Microfluidics enables the generation of uniform monodisperse droplets in a high-throughput manner, and these droplets are used as templates to fabricate polymeric microspheres with various physical and chemical properties. By co-loading monomers and photoinitiators in droplets and irradiating them with ultraviolet light for cross-linking polymerization, a large number of hydrogel microspheres can be obtained in a short time. The produced microspheres have uniform size, tunable pore size, good monodispersity and high yield. By changing the mass ratio of monomer to water in the droplet, hydrogel microspheres with different pore sizes can be prepared, which can be used for the selective detection of biological small molecules or metal ions in body fluids, achieving the goal of direct detection of biological fluids. Complex sample preparation steps are avoided.

目前，液滴水凝胶微球用于SERS检测还处于起步阶段，针对复杂的生物样本，仍需设计高灵敏、高特异性的生物传感器，实现小分子的快速精准检测。At present, the use of droplet hydrogel microspheres for SERS detection is still in its infancy. For complex biological samples, it is still necessary to design highly sensitive and specific biosensors to achieve rapid and accurate detection of small molecules.

发明内容Contents of the invention

本发明的目的是为了解决现有技术中存在的缺点，而提出的一种基于液滴水凝胶微球的SERS传感器的制备方法及其在血糖检测中的应用，该述SERS传感器具有高灵敏度和高特异性等特点，通过液滴水凝胶微球的引入解决了SERS检测中重复性差和需要复杂的样品前处理等问题，为SERS光谱技术在生物小分子检测中的应用开辟了新的策略。The purpose of the present invention is to solve the shortcomings in the prior art, and propose a method for preparing a SERS sensor based on droplet hydrogel microspheres and its application in blood glucose detection. The SERS sensor has high sensitivity and With the characteristics of high specificity, the introduction of droplet hydrogel microspheres solves the problems of poor repeatability and complex sample pretreatment in SERS detection, and opens up a new strategy for the application of SERS spectroscopy in the detection of small biological molecules.

为了实现上述目的，本发明采用了如下技术方案：In order to achieve the above object, the present invention adopts the following technical solutions:

一种基于液滴水凝胶微球的SERS传感器的制备方法，具体步骤如下：A method for preparing a SERS sensor based on droplet hydrogel microspheres, the specific steps are as follows:

S1、SERS纳米探针AgNPs@GOX的制备：将半胱胺酸溶液加入到硝酸银AgNO₃溶液中于室温下搅拌30min；之后，加入NaBH₄溶液，于室温下在暗处和明处依次各搅拌20min，得到带正电的银纳米粒子AgNPs；取一定量GOX溶液加入到上述制备好的带正电的银纳米粒子中，室温下静置反应24h获得SERS纳米探针AgNPs@GOX；S1. Preparation of SERS nanoprobe AgNPs@GOX: add cysteine solution to silver nitrate AgNO₃ solution and stir at room temperature for 30 min; then add NaBH₄ solution and stir at room temperature in the dark and in the light After 20 minutes, positively charged silver nanoparticles AgNPs were obtained; a certain amount of GOX solution was added to the above-mentioned positively charged silver nanoparticles, and the reaction was left at room temperature for 24 hours to obtain SERS nanoprobes AgNPs@GOX;

S2、传感微球的制备：将AgNPs@GOX、聚乙二醇-二丙烯酸酯PEGDA溶液和光引发剂BASF水溶液分别作为三个水相，含表面活性剂ABILEM90的十六烷作为油相，采用注射泵使得水相和油相分别以一定流速注入十字型微流控芯片通道中，在芯片通道的十字交汇处通过油相切割水相形成单分散液滴；将生成的液滴收集在玻璃瓶中，置于紫外光下照射5min，使液滴内部的单体和引发剂发生交联形成基于液滴水凝胶微球的SERS传感器。S2. Preparation of sensing microspheres: AgNPs@GOX, polyethylene glycol-diacrylate PEGDA solution, and photoinitiator BASF aqueous solution were used as three aqueous phases, and hexadecane containing surfactant ABILEM90 was used as an oil phase. The syringe pump allows the water phase and the oil phase to be injected into the cross-shaped microfluidic chip channel at a certain flow rate, and the oil phase cuts the water phase at the cross intersection of the chip channel to form monodisperse droplets; the generated droplets are collected in glass bottles In the experiment, put it under ultraviolet light for 5 min to cross-link the monomer and initiator inside the droplet to form a SERS sensor based on droplet hydrogel microspheres.

优选地，在步骤S1中，GOX溶液的pH为5.8，浓度为4mg/mL；GOX和AgNPs溶液的体积比是1:10。Preferably, in step S1, the pH of the GOX solution is 5.8, and the concentration is 4 mg/mL; the volume ratio of GOX and AgNPs solution is 1:10.

优选地，在步骤S1中，半胱胺酸、AgNO₃和NaBH₄溶液的浓度分别是213mM、1.42mM和10mM，半胱胺酸溶液和硝酸银溶液的体积比是1:100。Preferably, in step S1, the concentrations of cysteine, AgNO₃ and NaBH₄ solutions are 213 mM, 1.42 mM and 10 mM respectively, and the volume ratio of cysteine solution and silver nitrate solution is 1:100.

优选地，在步骤S2中，聚乙二醇-二丙烯酸酯PEGDA溶液和光引发剂BASF溶液的质量分数分别是72％和2.5％；水相和油相的流速比是1:8。Preferably, in step S2, the mass fractions of the polyethylene glycol-diacrylate PEGDA solution and the photoinitiator BASF solution are 72% and 2.5% respectively; the flow rate ratio of the water phase and the oil phase is 1:8.

优选地，在步骤S2中，所用紫外光的光功率密度是78mW/cm²。Preferably, in step S2, the optical power density of the ultraviolet light used is 78mW/cm² .

本发明还提供了一种采用上述制备方法制得的基于液滴水凝胶微球的SERS传感器在检测血糖中的应用。The present invention also provides an application of the SERS sensor based on the droplet hydrogel microsphere prepared by the above preparation method in detecting blood sugar.

优选地，取10μL待测血糖加入150μL水凝胶微球中，将该混合溶液置于室温中涡旋振荡20min，使葡萄糖充分进入水凝胶微球内部；再将混合溶液置于37℃水浴中15min使GOX与葡萄糖发生催化反应；采集反应后水凝胶微球的SERS光谱，将1343cm^-1处的SERS强度值代入线性回归方程y＝4839.2-153.3x，相关系数R²是0.929，即可得到待测样品的浓度。Preferably, 10 μL of blood glucose to be tested is added to 150 μL of hydrogel microspheres, and the mixed solution is vortexed at room temperature for 20 minutes to allow the glucose to fully enter the interior of the hydrogel microspheres; then the mixed solution is placed in a 37°C water bath GOX and glucose were catalyzed for 15 minutes; the SERS spectrum of the hydrogel microspheres after the reaction was collected, and the SERS intensity value at 1343 cm⁻¹ was substituted into the linear regression equation y=^{4839.2-153.3x} , and the correlation coefficient R2 was 0.929, namely The concentration of the sample to be tested can be obtained.

通过采用上述技术方案：将修饰有葡萄糖氧化酶(GOX)的银纳米粒子封装在孔状水凝胶微球里，血液中的小分子葡萄糖通过优化的孔选择性地进入微球内部，葡萄糖与GOX发生催化反应生成的葡萄糖酸刻蚀银纳米粒子，使得GOX的SERS信号减弱，根据GOX的SERS信号强度变化实现葡萄糖的检测。By adopting the above technical scheme: encapsulating silver nanoparticles modified with glucose oxidase (GOX) in porous hydrogel microspheres, the small molecule glucose in the blood selectively enters the interior of the microspheres through the optimized pores, and the glucose and The gluconic acid generated by the catalytic reaction of GOX etches the silver nanoparticles, which weakens the SERS signal of GOX, and the detection of glucose is realized according to the change of the SERS signal intensity of GOX.

与现有技术相比，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:

1、本发明利用液滴水凝胶微球为载体，将SERS纳米探针包覆于微球内部，结合酸性刻蚀纳米粒子导致其拉曼增强效果减弱原理，设计了一种快速、高灵敏度、高选择性直接检测血糖的生物传感器。1. The present invention uses droplet hydrogel microspheres as a carrier to coat SERS nanoprobes inside the microspheres, and combines the principle of weakening the Raman enhancement effect of the nanoparticles by acidic etching, and designs a fast, high-sensitivity, A biosensor for direct detection of blood glucose with high selectivity.

2、本发明制得的传感器中的水凝胶微球具有大小可调的孔径，能够排除血液中的大分子蛋白，只允许小分子葡萄糖选择性通过孔进入微球内部，实现了对复杂生物样品的直接定量SERS分析；水凝胶基质能够保护金属纳米粒子免受周围环境的污染，改善了SERS基底的化学稳定性；由于纳米粒子在水凝胶微球中的均匀分散，提高了SERS检测的重现性。2. The hydrogel microspheres in the sensor prepared by the present invention have an adjustable pore size, which can exclude macromolecular proteins in the blood, and only allow small molecule glucose to selectively enter the microsphere through the pores, realizing the detection of complex organisms. Direct quantitative SERS analysis of samples; the hydrogel matrix can protect the metal nanoparticles from the pollution of the surrounding environment, improving the chemical stability of the SERS substrate; due to the uniform dispersion of the nanoparticles in the hydrogel microspheres, the SERS detection is improved reproducibility.

附图说明Description of drawings

图1为本发明制备SERS传感器所用银纳米粒子的紫外吸收光谱及透射电子显微镜图；Fig. 1 is that the present invention prepares the ultraviolet absorption spectrum and the transmission electron micrograph of the used silver nanoparticle of SERS sensor;

图2为本发明实施例2中采用动态光散射仪测得的AgNPs和AgNPs@GOX的粒径及电位数据图；Fig. 2 is the particle size and potential data diagram of AgNPs and AgNPs@GOX measured by dynamic light scattering instrument in Example 2 of the present invention;

图3为本发明制备的水凝胶微球的明场显微镜照片(a)和扫描电子显微镜照片(b)；Fig. 3 is the bright field photomicrograph (a) and the scanning electron micrograph (b) of the hydrogel microsphere prepared by the present invention;

图4为对比水凝胶微球(1)、负载AgNPs@GOX的水凝胶微球在加入葡萄糖溶液前(3)和后(2)的SERS光谱；Figure 4 shows the SERS spectra of hydrogel microspheres (1) and AgNPs@GOX-loaded hydrogel microspheres before (3) and after (2) adding glucose solution;

图5中(a)为不同浓度血糖出现时GOX的SERS光谱图，(b)为GOX在1343cm^-1处SERS强度值与血糖浓度的关系图；Figure 5 (a) is the SERS spectrum of GOX when different concentrations of blood sugar appear, (b) is the relationship between the SERS intensity value of GOX at 1343cm^-1 and the blood sugar concentration;

图6为本发明GOX在1343cm^-1处SERS强度变化值与不同种类糖形成的柱状图；Fig. 6 is a histogram of the SERS intensity change value of GOX of the present invention at 1343 cm^-1 and different types of sugars;

图7为本发明同一个传感微球上不同位置处的SERS光谱图(选取任意的10个点)。Fig. 7 is a SERS spectrogram at different positions on the same sensing microsphere of the present invention (10 random points are selected).

具体实施方式detailed description

下面结合附图将对本发明实施例中的技术方案进行清楚、完整地描述，以使本领域的技术人员能够更好的理解本发明的优点和特征，从而对本发明的保护范围做出更为清楚的界定。本发明所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例，基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings, so that those skilled in the art can better understand the advantages and characteristics of the present invention, so as to make the protection scope of the present invention more clear definition. The embodiments described in the present invention are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without creative work For example, all belong to the protection scope of the present invention.

实施例1：基于液滴水凝胶微球的SERS传感器的构建，步骤如下：Example 1: The construction of a SERS sensor based on droplet hydrogel microspheres, the steps are as follows:

(1)将半胱胺酸溶液(400μL，213mM)加入到40mL浓度是1.42mM的AgNO₃溶液中。在室温下搅拌30min后，加入10μL浓度是10mM的NaBH₄溶液，室温下在暗处搅拌20min，然后在明处再搅拌20min，得到的灰绿色溶液放入冰箱(4℃)保存备用。取100μL浓度是4mg/mL的GOX溶液(pH＝5.8)加入到1mL上述制备好的带正电的银纳米粒子溶液中，室温下静置反应24h。多余的未修饰在AgNPs上的GOX通过离心(8000rpm，10min)去除，用去离子水洗涤离心后的沉淀物得到AgNPs@GOX。(1) Cysteine solution (400 μL, 213 mM) was added to 40 mL of AgNO₃ solution with a concentration of 1.42 mM. After stirring at room temperature for 30 min, 10 μL of₁₀ mM NaBH4 solution was added, stirred at room temperature for 20 min in the dark, and then stirred for another 20 min in the light, and the obtained gray-green solution was stored in the refrigerator (4°C) for later use. 100 μL of GOX solution (pH=5.8) with a concentration of 4 mg/mL was added to 1 mL of the above-prepared positively charged silver nanoparticle solution, and left to react at room temperature for 24 hours. The excess unmodified GOX on AgNPs was removed by centrifugation (8000rpm, 10min), and the centrifuged precipitate was washed with deionized water to obtain AgNPs@GOX.

(2)将AgNPs@GOX，质量分数是72％的聚乙二醇-二丙烯酸酯溶液和质量分数是2.5％的光引发剂(BASF)水溶液分别作为三个水相，采用注射泵使水相以35μL的流速通过内径0.8mm，外径1.6mm的聚乙烯管后注入到微流控芯片通道中，同样用注射泵以280μL的流速使含7％w/w表面活性剂(ABILEM90)的十六烷连续相注入芯片通道中，可以发现在通道的十字交汇处，油相切割水相形成了单分散液滴。将生成的液滴收集在玻璃瓶中，用紫外光(488nm，78mW/cm²)持续照射瓶中液滴，使液滴中的PEGDA发生交联形成水凝胶微球。然后用水洗涤三次，将所得的传感水凝胶微球置于水中保存。(2) AgNPs@GOX, polyethylene glycol-diacrylate solution with a mass fraction of 72% and a photoinitiator (BASF) aqueous solution with a mass fraction of 2.5% were used as three aqueous phases, and the aqueous phase was made by using a syringe pump. Pass through a polyethylene tube with an inner diameter of 0.8 mm and an outer diameter of 1.6 mm at a flow rate of 35 μL and then inject it into the channel of the microfluidic chip, and also use a syringe pump to make the 10% surfactant (ABILEM90) containing 7% w/w at a flow rate of 280 μL. The hexane continuous phase is injected into the channel of the chip, and it can be found that at the intersection of the channel, the oil phase cuts the water phase to form monodisperse droplets. The generated droplets were collected in a glass bottle, and the droplets in the bottle were continuously irradiated with ultraviolet light (488nm, 78mW/cm² ), so that the PEGDA in the droplets was cross-linked to form hydrogel microspheres. Then wash with water three times, and store the obtained sensing hydrogel microspheres in water.

实施例2：SERS纳米探针及液滴水凝胶微球的表征Example 2: Characterization of SERS nanoprobes and droplet hydrogel microspheres

首先对制备的纳米探针进行了表征。通过在AgNPs上修饰GOX获得SERS纳米探针。制备的AgNPs的紫外最大吸收波长是436nm(图1)，插图是AgNPs的透射电子显微镜照片，图像显示其尺寸约为40nm。为了表征SERS纳米探针是否成功制备，利用动态光散射仪测定了AgNPs和AgNPs@GOX的粒径及电位(图2)。单独的AgNPs的平均粒径是46.3nm，当修饰GOX之后，平均粒径增大到53.5nm，表明GOX被成功修饰在AgNPs上；AgNPs的电位是28.6mV，而AgNPs@GOX的电位降到了16.1mV，同样也证明了在AgNPs上成功修饰了GOX(呈负电)。Firstly, the prepared nanoprobes were characterized. SERS nanoprobes were obtained by modifying GOX on AgNPs. The maximum ultraviolet absorption wavelength of the prepared AgNPs is 436nm (Figure 1). The inset is a transmission electron micrograph of AgNPs, and the image shows that its size is about 40nm. In order to characterize whether the SERS nanoprobes were successfully prepared, the particle size and potential of AgNPs and AgNPs@GOX were measured by dynamic light scattering (Fig. 2). The average particle size of AgNPs alone is 46.3nm. After modifying GOX, the average particle size increased to 53.5nm, indicating that GOX was successfully modified on AgNPs; the potential of AgNPs was 28.6mV, while the potential of AgNPs@GOX dropped to 16.1 mV, also demonstrated the successful modification of GOX (negatively charged) on AgNPs.

对制备的液滴水凝胶微球进行形貌表征。图3(a)和(b)分别是水凝胶微球的明场显微镜照片和扫描电子显微镜照片，得到的水凝胶微球呈球形且尺寸均一(约为50μm)。The morphology of the prepared droplet hydrogel microspheres was characterized. Figure 3(a) and (b) are the bright-field micrographs and scanning electron micrographs of the hydrogel microspheres, respectively. The obtained hydrogel microspheres are spherical and uniform in size (about 50 μm).

实施例3：SERS传感器测定葡萄糖的原理可行性Embodiment 3: The principle feasibility of SERS sensor measuring glucose

分别采集水凝胶微球、负载AgNPs@GOX的水凝胶微球在加入葡萄糖溶液前后的SERS光谱。如图4所示，单独的水凝胶微球几乎没有特征的SERS谱峰出现，表明水凝胶微球自身没有SERS信号，不会干扰后续葡萄糖的测定。而负载AgNPs@GOX的水凝胶微球呈现很强的SERS光谱，当将其置于葡萄糖溶液中，SERS光谱的谱峰位置无变化但信号强度明显减弱。这是由于葡萄糖可以和GOX反应，生成过氧化氢(H₂O₂)，H₂O₂能够刻蚀银纳米粒子削弱其拉曼增强效果，导致GOX的SERS信号减弱。根据GOX的SERS信号强度变化实现葡萄糖的检测。The SERS spectra of hydrogel microspheres and AgNPs@GOX-loaded hydrogel microspheres were collected before and after adding glucose solution. As shown in Figure 4, almost no characteristic SERS spectrum peaks appear on the hydrogel microspheres alone, indicating that the hydrogel microspheres themselves have no SERS signal and will not interfere with the subsequent determination of glucose. While the hydrogel microspheres loaded with AgNPs@GOX exhibited a strong SERS spectrum, when it was placed in glucose solution, the peak position of the SERS spectrum did not change but the signal intensity was significantly weakened. This is because glucose can react with GOX to generate hydrogen peroxide (H₂ O₂ ), and H₂ O₂ can etch silver nanoparticles to weaken its Raman enhancement effect, resulting in the weakening of the SERS signal of GOX. The detection of glucose is realized according to the change of the SERS signal intensity of GOX.

实施例4：SERS传感器检测不同浓度的血糖Embodiment 4: SERS sensor detects blood sugar of different concentrations

首先将全血在室温下放置7h，使其发生糖酵解以消耗掉全血中的葡萄糖，然后采用加标方法测定不同浓度血糖。图5(a)是测得的不同浓度血糖下GOX的SERS光谱。随着血糖浓度的增加，SERS光谱的强度逐渐减弱。且在0-20mM区间内，血糖浓度与SERS强度值(1343cm^-1)呈现良好的线性关系。线性回归方程是y＝4839.2-153.3X，相关系数R²是0.929。采用该SERS传感器可以测定0-20mM范围内不同浓度的血糖且最低检测浓度是0.1mM。First, the whole blood was placed at room temperature for 7 hours to allow glycolysis to consume the glucose in the whole blood, and then the blood glucose at different concentrations was measured by the standard addition method. Figure 5(a) is the measured SERS spectra of GOX under different concentrations of blood glucose. With the increase of blood glucose concentration, the intensity of SERS spectrum gradually weakened. And in the range of 0-20mM, there is a good linear relationship between blood glucose concentration and SERS intensity value (1343cm^-1 ). The linear regression equation is y=^{4839.2-153.3X} , and the correlation coefficient R2 is 0.929. The SERS sensor can be used to measure different concentrations of blood sugar in the range of 0-20mM, and the lowest detection concentration is 0.1mM.

实施例5：评价检测特异性和重复性Example 5: Evaluation of detection specificity and repeatability

利用SERS光谱验证该方法测定血糖的特异性和重复性。为了评价该传感器的特异性，选取不同种类的糖(果糖、麦芽糖、蔗糖、半乳糖)以及目标物葡萄糖与该液滴水凝胶微球反应。测定相应的SERS光谱，并以SERS光谱中1343cm^-1处的强度值变化为纵坐标，不同种类的糖为横坐标作柱状图，结果如图6所示，相比其它糖类，葡萄糖的信号强度变化最明显，表明该SERS传感器对其他干扰物几乎没有响应，对葡萄糖有很高的选择性。The specificity and repeatability of the method for measuring blood glucose were verified by SERS spectrum. In order to evaluate the specificity of the sensor, different kinds of sugars (fructose, maltose, sucrose, galactose) and target glucose were selected to react with the droplet hydrogel microspheres. Measure the corresponding SERS spectrum, and use the intensity value change at 1343cm^-1 in the SERS spectrum as the ordinate, and different types of sugars as the abscissa to make a histogram. The results are shown in Figure 6. Compared with other sugars, the signal of glucose The intensity change is the most obvious, indicating that the SERS sensor has little response to other interfering substances and high selectivity to glucose.

为验证该SERS传感器的检测重复性，在同一个传感微球上不同位置处任取十个点，采集每个点的SERS光谱，将这些光谱统一作图。结果如图7所示，十个位置处SERS光谱的峰形和强度几乎没有变化，说明该传感器的重复性良好。In order to verify the detection repeatability of the SERS sensor, ten points were randomly selected at different positions on the same sensing microsphere, the SERS spectrum of each point was collected, and these spectra were uniformly plotted. The results are shown in Figure 7, the peak shapes and intensities of the SERS spectra at the ten positions hardly change, indicating that the sensor has good repeatability.

本发明中披露的说明和实践，对于本技术领域的普通技术人员来说，都是易于思考和理解的，且在不脱离本发明原理的前提下，还可以做出若干改进和润饰。因此，在不偏离本发明精神的基础上所做的修改或改进，也应视为本发明的保护范围。The description and practice disclosed in the present invention are easy to think and understand for those skilled in the art, and some improvements and modifications can be made without departing from the principle of the present invention. Therefore, modifications or improvements made on the basis of not departing from the spirit of the present invention should also be regarded as the protection scope of the present invention.

Claims (6)

Translated fromChinese

1.一种基于液滴水凝胶微球的SERS传感器的制备方法，其特征在于，具体步骤如下：1. A method for preparing a SERS sensor based on droplet hydrogel microspheres, characterized in that, the specific steps are as follows:S1、SERS纳米探针AgNPs@GOX的制备：将半胱胺酸溶液加入到硝酸银AgNO₃溶液中于室温下搅拌30min；之后，加入NaBH₄溶液，于室温下在暗处和明处依次各搅拌20min，得到带正电的银纳米粒子AgNPs；取一定量GOX溶液加入到上述制备好的带正电的银纳米粒子中，室温下静置反应24h获得SERS纳米探针AgNPs@GOX；S1. Preparation of SERS nanoprobe AgNPs@GOX: add cysteine solution to silver nitrate AgNO₃ solution and stir at room temperature for 30 min; then add NaBH₄ solution and stir at room temperature in the dark and in the light After 20 minutes, positively charged silver nanoparticles AgNPs were obtained; a certain amount of GOX solution was added to the above-mentioned positively charged silver nanoparticles, and the reaction was left at room temperature for 24 hours to obtain SERS nanoprobes AgNPs@GOX;S2、传感微球的制备：将AgNPs@GOX、聚乙二醇-二丙烯酸酯PEGDA溶液和光引发剂BASF水溶液分别作为三个水相，含表面活性剂ABIL EM90的十六烷作为油相，采用注射泵使得水相和油相分别以一定流速注入十字型微流控芯片通道中，在芯片通道的十字交汇处通过油相切割水相形成单分散液滴；将生成的液滴收集在玻璃瓶中，置于紫外光下照射5min，使液滴内部的单体和引发剂发生交联形成基于液滴水凝胶微球的SERS传感器。S2. Preparation of sensing microspheres: AgNPs@GOX, polyethylene glycol-diacrylate PEGDA solution, and photoinitiator BASF aqueous solution were used as three aqueous phases, and hexadecane containing surfactant ABIL EM90 was used as an oil phase. A syringe pump is used to inject the water phase and the oil phase into the cross-shaped microfluidic chip channel at a certain flow rate, and the oil phase cuts the water phase at the cross intersection of the chip channel to form monodisperse droplets; the generated droplets are collected on the glass. Put it in the bottle and irradiate it with ultraviolet light for 5 minutes to cross-link the monomer and initiator inside the droplet to form a SERS sensor based on droplet hydrogel microspheres.2.根据权利要求1所述的一种基于液滴水凝胶微球的SERS传感器的制备方法，其特征在于，在步骤S1中，GOX溶液的pH为5.8，浓度为4mg/mL；GOX和AgNPs溶液的体积比是1:10。2. A method for preparing a SERS sensor based on droplet hydrogel microspheres according to claim 1, characterized in that, in step S1, the pH of the GOX solution is 5.8, and the concentration is 4mg/mL; GOX and AgNPs The volume ratio of the solution is 1:10.3.根据权利要求1所述的一种基于液滴水凝胶微球的SERS传感器的制备方法，其特征在于，在步骤S2中，聚乙二醇-二丙烯酸酯PEGDA溶液和光引发剂BASF溶液的质量分数分别是72％和2.5％；水相和油相的流速比是1:8。3. the preparation method of a kind of SERS sensor based on droplet hydrogel microspheres according to claim 1, is characterized in that, in step S2, polyethylene glycol-diacrylate PEGDA solution and photoinitiator BASF solution The mass fractions are 72% and 2.5% respectively; the flow rate ratio of water phase and oil phase is 1:8.4.根据权利要求1所述的一种基于液滴水凝胶微球的SERS传感器的制备方法，其特征在于，在步骤S2中，所用紫外光的光功率密度是78mW/cm²。4 . The method for preparing a SERS sensor based on droplet hydrogel microspheres according to claim 1 , wherein in step S2 , the optical power density of the ultraviolet light used is 78 mW/cm² .5.一种采用权利要求1-5任一项所述的制备方法制得的基于液滴水凝胶微球的SERS传感器在检测血糖中的应用。5. An application of a SERS sensor based on droplet hydrogel microspheres prepared by the preparation method according to any one of claims 1-5 in detecting blood sugar.6.根据权利要求6所述的应用，其特征在于，取10μL待测血糖加入150μL水凝胶微球中，将该混合溶液置于室温中涡旋振荡20min使葡萄糖充分进入水凝胶微球内部；再将混合溶液置于37℃水浴中15min使GOX与葡萄糖发生催化反应；采集反应后水凝胶微球的SERS光谱，将1343cm^-1处的SERS强度值代入线性回归方程y＝4839.2-153.3x，相关系数R²是0.929，即可得到待测样品的浓度。6. The application according to claim 6, wherein 10 μL of blood glucose to be tested is added to 150 μL of hydrogel microspheres, and the mixed solution is placed at room temperature and vortexed for 20 minutes to allow the glucose to fully enter the hydrogel microspheres Inside; put the mixed solution in a water bath at 37°C for 15 minutes to catalyze the reaction between GOX and glucose; collect the SERS spectrum of the hydrogel microspheres after the reaction, and substitute the SERS intensity value at 1343cm^-1 into the linear regression equation y=4839.2- 153.3x, and the correlation coefficient R² is 0.929, the concentration of the sample to be tested can be obtained.

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