A novel method to prepare SPR sensor chips based on photografting molecularly imprinted polymer

A novel method to prepare surface plasmon resonance(SPR) sensor chips based on grafted imprinted polymer is explored. Benzophenone photografting system is used to grow molecularly imprinted polymer(MIP) films from the modified surface of gold substrate.The surface morphology and thickness of MIP films were investigated by scanning electronic microscope(SEM).The adsorption properties of sensor chip were studied by SPR spectroscopy.The results demonstrate that nano-MIP films can be constructed on the surface of gold substrate with the good adsorption of template molecules.     

Ultrasensitively sensing acephate using molecular imprinting techniques on a surface plasmon resonance sensor

An ultrathin molecularly imprinted polymer film was anchored on an Au surface for fabricating a surface plasmon resonance sensor sensitive to acephate by a surface-bound photo-radical initiator. The polymerization in the presence of acephate resulted in a molecular-imprinted matrix for the enhanced binding of acephate. Analysis of the SPR wavenumber changes in the presence of different concentrations of acephate gave a calibration curve that included the ultrasensitive detection of acephate by the imprinted sites in the composite, K_ass for the association of acephate to the imprinted sites, 7.7 × 10¹² M⁻¹. The imprinted ultrathin film revealed impressive selectivity. The selectivity efficiencies for acephate and other structurally related analogues were 1.0 and 0.11-0.37, respectively. Based on a signal to noise ratio of 3, the detection limits were 1.14 × 10⁻¹³ M for apple sample and 4.29 × 10⁻¹⁴ M for cole sample. The method showed good recoveries and precision for the apple and cole samples spiked with acephate solution. This suggests that a combination of SPR sensing with MIP film is a promising alternative method for the detection of organophosphate compounds.     

表面接枝法制备睾酮素分子印迹表面等离子体共振传感器

构建了一种选择性检测睾酮素的分子印迹表面等离子体共振( Surface plasmon resonance, SPR)传感器。采用紫外光引发表面接枝技术,在固定引发转移终止剂的SPR芯片表面制备了以睾酮素为模板分子,甲基丙烯酸为功能单体,乙二醇二甲基丙烯酸酯为交联剂的分子印迹膜( Molecularly imprinted film, MIF)。利用SPR在目标共振角处现场监测使MIF聚合成膜过程更易控。偏振调制-红外反射吸收光谱表征证明MIF接枝成功。原子力显微镜结果显示,MIF 表面均匀散布着纳米尺寸的孔穴。利用 SPR 对2.5×10-16~2.5×10-6 mol/L睾酮素进行吸附检测,检出限低至2.5×10-16 mol/L,对低浓度和高浓度的睾酮素吸附分段进行线性拟合,得到拟合线性方程分别为y=19.69+1.21x(R2=0.9913)和y=11.5+0.45x(R2=0.9895);睾酮素类似物雌二醇、雌三醇和黄体酮的吸附实验结果显示,此印迹膜对模板分子有很好的选择性;5次重复洗脱吸附后,MIF仍保持较好的传感性能,说明此传感器具有较高的稳定性和重复利用性;在人工尿液样品中测得的睾酮素回收率为85.2%~92.8%,说明此传感器可以用于实际样品测定。     

A 3,3′-dichlorobenzidine-imprinted polymer gel surface plasmon resonance sensor based on template-responsive shrinkage

Molecularly imprinted polymer gel film on the gold substrate of a chip was prepared with minute amount of cross-linker for the fabrication of a surface plasmon resonance (SPR) sensor sensitive to 3,3′-dichlorobenzidine. The molecularly imprinted gel film was anchored on a gold chip by a surface-bound photo-radical initiator. The sensing of 3,3′-dichlorobenzidine is based on responsive shrinkage of the imprinted polymer gel film that is triggered by target binding. This change can improve the responsiveness of the imprinted SPR sensor to 3,3′-dichlorobenzidine. The molecularly imprinted polymer gel film was characterized with contact angle measurements, electrochemical impedance spectroscopy, cyclic voltammogram, swelling measurements and atomic force microscopy. The changes of SPR spectroscopy wavenumber shifts revealed that the imprinted gel sensing film can ‘memorize’ the binding of 3,3′-dichlorobenzidine compared to non-imprinted one. The imprinted gel-SPR sensor showed a linear response in the range of 9.0 × 10⁻¹² to 5.0 × 10⁻¹⁰ mol L⁻¹ (R² = 0.9998) for the detection of 3,3′-dichlorobenzidine, and it also exhibited high selectivity to 3,3′-dichlorobenzidine compared to its structurally related analogues. We calculated the detection limits to be 0.471 ng L⁻¹ for tap water and 0.772 ng kg⁻¹ for soil based on a signal to noise ratio of 3. The method showed good recoveries and precision for the samples spiked with 3,3′-dichlorobenzidine. This suggest that the imprinted gel-SPR sensing method can be used as a promising alternative for the detection of 3,3′-dichlorobenzidine.     

Molecularly Imprinted Polymer Thin Film Based Surface Plasmon Resonance Sensor to Detect Hemoglobin

Molecularly imprinted polymer（MIP） films for hemoglobin detection were prepared onto the Au/Cr coated surface plasmon resonance（SPR） sensor chips by the in situ electropolymerization of 3-aminophenylboronic acid（3-APBA）.The formation of the films and rebinding processes of hemoglobin were monitored by in situ electrochemical-SPR（EC-SPR） spectroscopy,with allowed real-time observation of the simultaneous changes in electrochemical and optical properties of the films.Scanning electron microscopy（SEM） and atomic force microscopy（AFM）were used to characterize the surface morphologies of the MIP films.The effects of pH,ion strength,different metal ions on rebinding Hb,the specific binding and the selective recognition were investigated.The results obtained with the molecular imprinted SPR chips indicate a good adsorption of Hb in a range of 0.0005-5 mg/mL in 0.05 mol/L sodium phosphate buffer at pH=7.0.A linear calibration curve（R2=0.94） of the SPR sensor for Hb detection was obtained in a range of 0.05-5 mg/mL.The detection limit for hemoglobin by this method was 0.000435 mg/mL（S/N=3）.Interference studies indicate that the MIP films have a good selectivity compared with the referenced proteins.The stability of the sensor was also established.Results indicate that the SPR sensor chip keeps 87.6％ of its original response after 14 d of storage under dry and ambient conditions.     

Detection of parathion methyl using a surface plasmon resonance sensor combined with molecularly imprinted films

An ultra-sensitive and highly selective parathion methyl(PM) detection method by surface plasmon resonance(SPR) combined with molecularly imprinted films(MIF) was developed. The PM-imprinted film was prepared by thermo initiated polymerization on the bare Au surface of an SPR sensor chip.Template PM molecules were quickly removed by an organic solution of acetonitrile/acetic acid(9:1,v/v), causing a shift of 0.58 in SPR angle. In the concentrations range of 10à13–10à10mol/L, the refractive index showed a gradual increase with higher concentrations of template PM and the changes of SPR angles were linear with the negative logarithm of PM concentrations. In the experiment, the minimum detectable concentration was 10à13mol/L. The selectivity of the thin PM-imprinted film against diuron,tetrachlorvinphose and fenitrothion was examined, but no observable binding was detected. The results in the experiment suggested that the MIF had the advantages of high sensitivity and selectivity.     

热聚合制备分子印迹膜的表面等离子体共振现场监测方法

本研究组设计了SPR现场监测MIFs热聚合成膜过程的装置, 并对所制备MIFs的吸附特性进行了检测.     

Real-time analysis of enzymatic surface-initiated polymerization using surface plasmon resonance (SPR)

The kinetics of enzymatic surface-initiated polymerization of PHB on gold surface has been examined by SPR and the resultant polymer layers characterized by AFM and FT-IR spectrometry. The immobilized enzyme catalyzed surface-initiated polymerization of 3HB-CoA, resulting in the formation of a polymer brush on the surface. The rate of polymer growth from the surface was monitored by SPR in real-time. Polymer growth as measured by the increase in the resonance angle showed no apparent lag phase during the polymerization reaction. SPR analysis also revealed that the thickness of the polymer film could be controlled by varying the initial enzyme density on the surface. The average thicknesses of the PHB film after polymerization reaction were 95, 45 and 15 nm for the surfaces that were treated with 0.5, 0.3 and 0.1*10(-6) M of enzyme, respectively. The binding of PHA synthase at different concentration to the mixed SAMs and subsequent polymerization.     

苏丹红Ⅰ印迹表面等离子共振传感器的制备与表征

以苏丹红I印迹的凝胶膜对模板分子的响应收缩为基础,利用表面引发聚合技术在表面等离子共振（SPR）芯片上制备了苏丹红I印迹SPR传感器,利用电化学和溶胀测量技术对传感器进行表征,建立了对苏丹红I的SPR检测方法。该方法的线性范围为8.0×10-10~1.0×10-8 mol/L(R2=0.9987)。 方法用于样品红辣椒粉和腌制品中苏丹红I的检测,其检出限分别为3.4×10-10和2.5×10-10 mol/L。 红辣椒粉中两个样品的相对标准偏差为1.38%和2.10%,腌制品试样的相对标准偏差为1.13%。 说明该方法具有较高的灵敏度和较好的的重现性。     

Surface plasmon resonance sensor chips for the recognition of bovine serum albumin via electropolymerized molecularly imprinted polymers

In this paper,a surface plasmon resonance(SPR)sensor chip for detection of bovine serum album(BSA)was prepared by electropolymerization of 3-aminophenylboronic acid(3-APBA)based on molecularly imprinted polymer(MIP)technique.The surface morphology of MIP and non-imprinted(NIP)flms were characterized by scanning electroscopy(SEM).SEM images exhibited nanoscale cavities formed on the MIP films surface homogeneously due to the removal of BSA templates.The effects of pH,ion strength of rebinding BSA,the specific binding and selective recognition were studied for MIP films.Results indicated that the BSA-imprinted films exhibited a good adsorption of template protein(0.02–0.8 mg/mL)in0.05 mol/L sodium phosphate buffer at pH 5.0 with the limit of detection(LOD)of 0.02 mg/mL.     

间接抑制免疫分析法测定水溶液中痕量磺胺甲噁唑

将分子印迹技术与表面等离子体共振技术联用,建立了一种新型间接抑制免疫分析法,并用于磺胺甲唑的测定.采用管内原位聚合的方法,在长30 cm,内径0.25 mm的毛细管内制备了磺胺甲唑印迹聚合物涂层.涂层厚度由扫描电子显微镜测得为198 nm.在BIAcore 3000生物传感器上自动进行检测.涂层毛细管用于免疫检测前的在线固相萃取和预富集.磺胺甲唑单克隆抗体被富集的磺胺甲唑抑制,抑制信号与磺胺甲唑的浓度成正比.在优化的实验条件下,方法的线性范围为0.04～10.0 μg/L; 检出限为0.01 μg/L.本方法可直接用于实际样品的检测,回收率好,灵敏度高,操作简便,自动化程度高.     

Electrochemical sensors based on molecularly imprinted polymers grafted onto gold electrodes using click chemistry

We have developed a three-step method to graft molecularly imprinted polymer (MIP) thin films onto Au electrodes. In the first step, propargyl acrylate is clicked onto an azidoundecanethiol (N(3)(CH(2))(11)SH)/decanethiol mixed self-assembled monolayer (SAM). Then, by applying UV light (365 nm) in the presence of N,N'-methylenebis(acrylamide) (MAAM) and azobisisobutyronitrile (AIBN) as the radical initiator, polymerization was carried out directly on the electrode surface in the presence of an electroactive template molecule, hydroquinone (HQ). Detection of HQ using the clicked-on MIP sensor was studied using chronoamperometry and its behavior was compared to that of a sensor prepared by drop-coating MIPs onto Au. The detection limit of the clicked-on MIP sensor for HQ was found to be 1.21±0.56 μM, about four times lower than what was observed using the coated-on MIP sensor. In addition, the sensitivity of the clicked-on MIP sensor was found to be approximately three times greater than the coated-on MIP sensor. Apparent diffusion coefficients determined using chronoamperometry suggest that the improved performance is likely due to the favorable mass transfer characteristics of the clicked-on MIP sensing membrane.     

Achieving highly effective nonfouling performance for surface-grafted poly(HPMA) via atom-transfer radical polymerization

Human blood plasma and serum pose significant challenges to implanted devices because of highly unfavorable nonspecific protein adsorption on the surface. In this work, we introduce an improved two-step method to immobilize initiator thiols on a gold substrate for the surface-initiated atom-transfer radical polymerization (SI-ATRP) of hydroxypropyl methacrylate (HPMA). We investigate protein adsorption from a single-protein solution, diluted (10%) and undiluted (100%) human blood plasma, and serum on the poly(HPMA) brushes with different film thicknesses using surface plasmon resonance (SPR) sensors. SPR results show a correlation between antifouling properties and film thickness; that is, the poly(HPMA) brushes exhibit high protein resistance at medium film thicknesses of ～25-40 nm (e.g. <0.3 ng/cm(2) for single-protein adsorption and 10% human blood plasma and serum, ～24.5 ng/cm(2) for 100% human serum, and ～52.8 ng/cm(2) for 100% human plasma at a thickness of ～29 nm). With an optimal film thickness and surface roughness, the poly(HPMA) brush also demonstrates its high resistance to fibroblast adhesion. This work provides an alternative surface polymerization approach to preparing effective antifouling poly(HPMA) materials for potential applications in blood-contacting medical devices.     

Continuous Immunoassay for Sulfamethazine by Surface Plasmon Resonance-Based Biosensor

Regeneration of the sensor chip surface is difficult in many surface plasmon resonance (SPR) biosensor assays. Improper regeneration will reduce life span of the sensor chip and decrease the quality of the data. Considering the advantages of reducing the regeneration frequency, a theoretically feasible continuous SPR biosensor immunoassay for sulfamethazine (SMT) was developed. In the continuous inhibitive immunoassay, the sensor chip surface is regenerated only once after a definite number of tests instead of every test. The SMT-bovine serum albumin (BSA) conjugate was covalently immobilized to a carboxymethyldextran modified gold film. The immobilization conditions of the antigen were studied and the working dilution of the antibody was optimized. The antibody was mixed with SMT of different concentrations prepared with PBS buffer to construct the calibration curve. The limit of detection was 0.5 ng mL^?1. The continuous SPR biosensor assay was proved to be simpler and more practical than a normal one.     

Capacitive Chemical Sensor for Thiopental Assay Based on Electropolymerized Molecularly Imprinted Polymer

A novel capacitive sensor based on electropolymerized molecularly imprinted polymer (MIP) for thiopental detection is described. The molecularly imprinted film as a recognition element was prepared by electropolymerization of phenol on a gold electrode in the presence of thiopental (template). Cyclic voltammetry and capacitive measurements were used for characterization and evaluation of the polymeric film. The template molecules were removed from the modified electrode surface by washing with an ethanol:water solution. The sensor’s linear response range was between 3 and 20 µM, with a detection limit of 0.6 µM. The proposed sensor exhibited good selectivity, reproducibility. Satisfactory results were obtained in the direct detection of real samples.     

纳米多孔金薄膜的表面等离子体共振传感特性

对淀积在玻璃衬底上厚度约60 nm的金银合金溅射薄膜进行硝酸腐蚀脱银处理, 得到纳米多孔金薄膜. 利用自建的波长检测型表面等离子体共振(SPR)传感装置研究了腐蚀时间对纳米多孔金薄膜SPR特性的影响, 结果发现纳米多孔金薄膜与水溶液接触后在400-900 nm光谱范围内不具有SPR效应, 而当薄膜置于空气中时会产生明显的传播等离子体共振吸收峰, 其共振波长随腐蚀时间增加逐渐红移. 纳米多孔金薄膜在空气气氛中的SPR效应使其能够用于原位监测气相分子在孔内的吸附, 还可对在液相中吸附的生化分子进行离位测试. 本文对L-谷胱甘肽、L-半胱氨酸、2-氨基乙硫醇三种含巯基的生化小分子在纳米多孔金薄膜内的吸附进行了离位分析, 结果表明与传统的致密金薄膜SPR芯片比较, 纳米多孔金薄膜对这些分子显示出更高的灵敏度和更低的检测下限, 这归功于多孔金的大比表面积使其能够吸附大量的生化小分子. 实验还对乙醇蒸气在纳米多孔金薄膜内的吸附进行了原位监测, 发现吸附平衡所用时间较长, 约为160 min.     

基于分子印迹技术的丙溴磷压电石英晶体微天平研制

介绍了一种用于检测丙溴磷农药的分子印迹压电生物传感器的构建方法。采用沉淀聚合法合成了农药丙溴磷的分子印迹聚合物,将其固定于石英晶体微天平电极表面构建传感器;采用环境扫描电镜以及原子力显微镜对聚合物形貌、传感器电极表面形貌特征进行分析,并利用传感器对丙溴磷农药进行检测分析,其质量浓度在10~1000 ng/mL范围内,传感器频率改变与丙溴磷浓度之间的响应呈线性关系,线性方程为y=0.139ρ+2.26(r=0.9984)。结果表明,构建的分子印迹压电生物传感器能够对农药进行初步检测,具有较高的灵敏性和较好的特异识别能力。     

Acetylsalicylic acid electrochemical sensor based on PATP-AuNPs modified molecularly imprinted polymer film

A novel electrochemical sensor based on molecularly imprinted polymer film has been developed for aspirin detection. The sensitive film was prepared by co-polymerization of p-aminothiophenol (p-ATP) and HAuCl(4) on the Au electrode surface. First, p-ATP was self-assembled on the Au electrode surface by the formation of Au-S bonds. Then, the acetylsalicylic acid (ASA) template was assembled onto the monolayer of p-ATP through the hydrogen-bonding interaction between amino group (p-ATP) and oxygen (ASA). Finally, a conductive hybrid membrane was fabricated at the surface of Au electrode by the co-polymerization in the mixing solution containing additional p-ATP, HAuCl(4) and ASA template. Meanwhile, the ASA was spontaneously imprinted into the poly-aminothiophenol gold nanoparticles (PATP-AuNPs) complex film. The amount of imprinted sites at the PATP-AuNPs film significantly increases due to the additional replenishment of ASA templates. With the significant increasing of imprinted sites and doped gold nanoparticles, the sensitivity of the molecular imprinted polymer (MIP) electrode gradually increased. The molecularly imprinted sensor was characterized by electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV), and cyclic voltammetry (CV). The linear relationships between current and logarithmic concentration were obtained in the range from 1 nmol L(-1) to 0.1 μmol L(-1) and 0.7 μmol L(-1) to 0.1 mmol L(-1). The detection limit of 0.3 nmol L(-1) was achieved. This molecularly imprinted sensor for the determination of ASA has high sensitivity, good selectivity and reproducibility, with the testing in some biological fluids also has good selectivity and recovery.     

Surface plasmon resonance sensor for lysozyme based on molecularly imprinted thin films

Molecularly imprinted polymers (MIPs) selective for lysozyme were prepared on SPR sensor chips by radical co-polymerization with acrylic acid and N,N′-methylenebisacrylamide. Gold-coated SPR sensor chips were modified with N,N′-bis(acryloyl)cystamine, on which MIP thin films were covalently conjugated. The presence of NaCl during the polymerization and the re-binding tests affected the selectivity and the optimization of NaCl concentration in the pre-polymerization mixture and the re-binding buffer could enhance the selectivity in the target protein sensing. When the lysozyme-imprinted polymer thin films were prepared in the presence of 40 mM NaCl, the selectivity factor (target protein bound/reference protein bound) of MIP in the re-binding buffer containing 20 mM NaCl was 9.8, meanwhile, that of MIP in the re-binding buffer without NaCl was 1.2. A combination of SPR sensing technology with protein-imprinted thin films is a promising tool for the construction of selective protein sensors.     

Preparation of MIP grafts for quercetin by tandem aryl diazonium surface chemistry and photopolymerization

The food antioxidant quercetin was used as a template in an ultrathin molecularly imprinted polymer (MIP) film prepared by photopolymerization. Indium tin oxide (ITO) plates were electrografted with aryl layers via a diazonium salt precursor bearing two terminal hydroxyethyl groups. The latter act as hydrogen donors for the photosensitizer isopropylthioxanthone and enabled the preparation of MIP grafts through radical photopolymerization of methacrylic acid (the functional monomer) and ethylene glycol dimethacrylate (the crosslinker) in the presence of quercetin (the template) on the ITO. The template was extracted, and the remaining ITO electrode used for the amperometric determination of quercetin at a working potential of 0.26 V (vs. SCE). The analytical range is from 5.10^?8 to 10^?4 mol L^?1, and the detection limit is 5.10^?8 mol L^?1.