Surface plasmon resonance immunosensor for cortisol and cortisone determination

In this paper, we present a surface-plasmon-resonance-based immunosensor for the real-time detection of cortisol and cortisone levels in urine and saliva samples. The method proposed here is simple, rapid, economic, sensitive, robust, and reproducible thanks also to the special features of the polycarboxylate-hydrogel-based coatings used for the antibody immobilization. The sensor surface displays a high level of stability during repeated regeneration and affinity reaction cycles. The immunosensor shows high specificity for cortisol and cortisone; furthermore, no significant interferences from other steroids with a similar chemical structure have been observed. The suitability of the hydrogel coating for the prevention of nonspecific binding is also investigated. A good correlation is noticed between the results obtained by the proposed method and the reference liquid chromatography/tandem mass spectrometry method for the analysis of cortisol and cortisone in urine and saliva samples. Standard curves for the detection of cortisol and cortisone in saliva and urine are characterized by a detection limit less than 10 μg l⁻¹, sufficiently sensitive for both clinical and forensic use. Application of a newly developed SPR immunosensor for the measurement of cortisol in anti-doping analysis     

Immobilization of Escherichia coli for detection of phage T4 using surface plasmon resonance

Phage contamination is a very serious and unavoidable problem in modern fermentation industry.It is necessary to develop sensitive and rapid phage detection methods for the early detection of phage contamination.In the present work,a real-time,rapid,specific and quantitative phage T4 detection method based on surface plasmon resonance(SPR) technique has been introduced.Escherichia coli was immobilized onto the preformed MPA self-assembled monolayer(SAM) through the widely used EDC/NHS cross-linking reaction as the recognition element.The bacteria immobilization was verified efficiently through the electrochemical measurements and fluorescence microscopy observations.The specific adsorption was much stronger than the non-specific adsorption of phage T4 binding to the biosensor surface modified by E.coli,and the latter could be neglected.The detection sensitivity reached 1×10 7 PFU/mL within 10 min.Within the experimental phage concentrations,the linear correlation between the SPR response and the phage concentration was good.The results suggest that the SPR technique is a potentially powerful tool for the phage or other virus detections,as a label-free,real-time,and rapid method.     

Detection of Wild-Type Hypoxanthine Guanine Phosphoribosyl Transferase of Lymphocytes in Gamma-Irradiated Mice with Surface Plasmon Resonance

A quick and sensitive detection of the wild-type hypoxanthine guanine phosphoribosyl transferase (HPRT), which is known as a biomarker for radiation exposure, was developed. The conventional HPRT measurement technique is to detect the mutant HPRT, which is time-consuming and has low sensitivity. In this study, the wild-type HPRT was detected as a gamma radiation biomarker using a surface plasmon resonance (SPR) biosensor with 6-thioguanine (6-TG) as a probe and the anti-HPRT antibody as a signal amplification factor. First, we used this system to measure the wild-type HPRT dissolved in PBS. Six-TG immobilized on the surface can specifically detect the wild-type HPRT, and the anti-HPRT antibody enhances about 10 times the primary signal produced by the binding of the wild-type HPRT with 6-TG. A linear relationship (r = 0.991) was obtained between the concentration of the wild-type HPRT and the enhanced signal. The low detection limit (LDL) is 2.1 ng/mL. The regeneration using glycine-HCl was also investigated. Six-TG immobilized on the surface can be used in 9 injection-regeneration cycles. The relative standard deviation (RSD) of baseline changes after regenerations is 2.8%. The applicability of the method to real samples has been demonstrated with comparative analyses of lymphocyte extracts in gamma irradiated and unirradiated mice. The irradiated sample displays a significantly lower level of the wild-type HPRT compared to that in the unirradiated sample. The single sample detection only needs about 20 min. Thus, the SPR biosensor could potentially serve as an attractive technique for rapid and sensitive detection of the wild-type HPRT, a gamma radiation biomarker.     

Determination of Lysozyme by Thiol-Terminated Aptamer-Based Surface Plasmon Resonance

A sensitive and selective surface plasmon resonance (SPR) aptasensor has been developed for the real-time determination of lysozyme. The thiol-terminated lysozyme aptamer was covalently attached to the surface of sensor through Au–S bonding. In the presence of lysozyme, the aptamer captured lysozyme on the surface and enhanced the SPR signal that was proportional to the concentration of lysozyme. Under the optimized conditions, the SPR signal was linear with lysozyme concentration from 1 to 100?µmol/L. The detection limit for lysozyme was 0.5?µmol/L. The aptasensor also provided high specificity for lysozyme and was unaffected by other proteins. This aptasensor provides rapid, simple, and sensitive determination of lysozyme detection and a promising strategy for other proteins.     

Preparation of latex reagents combined with IgM and its F(ab')2 fragment from commercial ABO blood grouping reagent

To achieve a rapid assay for ABO blood grouping using a latex reagent, two latex reagents were produced, one of which combined with mouse monoclonal immunoglobulin M (IgM) isolated from commercial ABO blood grouping reagent, and the other of which combined with its F(ab')2 fragment prepared by cold pepsin digestion. The latex reagent adsorbing the F(ab')2 fragment was able to detect the 1000-fold diluted saliva and provided much better sensitivity than that of IgM. This suggests that the difference in sensitivity between the two latex reagents is responsible for adsorption orientation of the antigen site on the latex particles. The new assay successfully completed the ABO blood grouping of cigarette ends within 30 min.     

Surface Plasmon Resonance Coupled with Potential‐step Chronoamperometry: Theory and Applications for Quantitative Measurements of Electrodeposited Thin Films

Cyclic voltammetry (CV) has been combined with surface plasmon resonance (SPR) for probing electrochemical deposition and redox‐initiated film reorganization and conformational changes. However, the varying potential during CV scans leads to unwanted SPR background changes and complicates interpretation of SPR signals. In this work, we show that, when SPR is coupled with CV, the background correction for underpotential deposition of copper and electropolymerization of aniline is either inaccurate or difficult to perform. For accurate thickness measurements of electrodeposited films, potential‐step (PS) chronoamperometry is a method of choice to combine with SPR. The theory that interprets double‐layer charging is used to explain the advantage of PS chronoamperometry over CV in quantifying the thickness of electrodeposited thin films. The influence of the double‐layer charging on the potential‐induced SPR signal change was analyzed, and the results were used to optimize experimental parameters for PS‐SPR. Overall, PS‐SPR is easier to operate, simpler in data interpretation, and more accurate for the film thickness measurement.     

Highly sensitive assay for alkaline and acid phosphatase activities by high-performance liquid chromatography with electrochemical detection

A highly sensitive and specific assay for alkaline and acid phosphatases in biological materials, such as plasma and saliva, has been established. Phenol, formed enzymatically from the substrate phenylphosphate, was determined by high-performance liquid chromatography with electrochemical detection. The retention time of phenol was 7 min and no other peaks were observed. The method is rapid and sensitive with a detection limit for phenol of as little as 5 pmol. Thus, as little as 0.5 microliter of rat plasma or 10 microliters of human saliva is required for both alkaline and acid phosphatase assays. The assay is accurate and reproducible. Using this assay, alkaline and acid phosphatase activities in saliva were found to be 1.12 +/- 0.12 nmol/min/ml and 9.79 +/- 1.23 nmol/min/ml, respectively. This new assay method should be applicable to extremely small biological samples.     

Analysis of immunoarrays using a gold grating-based dual mode surface plasmon-coupled emission (SPCE) sensor chip

We have developed a novel dual mode immunoassay platform that combines the advantages of real-time, label free measurement of surface plasmon resonance (SPR) and the highly directional surface plasmon-coupled emission (SPCE) using a gold grating-based sensor chip. Since only fluorophore-labeled analyte molecules that are close to the metal surface of the sensor chip will couple to the surface plasmon, SPCE detection is highly surface-specific leading to background suppression and increased sensitivity. Theoretical calculations were done to find SPR and SPCE angles for a sensor chip optimized for Alexa Fluor 647. We have confirmed the SPR and SPCE responses on the dual mode sensor chip using Alexa Fluor 647 labeled anti-mouse IgG. Signal fluctuation of the dual mode sensor chip reader was below 1.2% and 0.8% for SPR and SPCE, respectively. The SPR response in this configuration showed a minimum detection level of 1 μg ml(-1), and the SPCE response showed a minimum detection level of 1 ng ml(-1) for the same sample. A range of human IgG concentrations in human serum was also analyzed with the dual mode sensor chip. The SPCE measurement is more sensitive than the SPR real-time measurement, and substantially extends the dynamic range of the assay platform, as well as enabling independent measurements of co-localized analytes on the same sensor chip region of interest. Since this assay platform is capable of measuring more than 1000 spatially encoded regions of interest on a 1 cm(2) sensor chip, it has the potential for high-content analyses of biological samples with both research and clinical applications.     

Simultaneous quantitation of salivary carbamazepine, carbamazepine-10,11-epoxide, phenytoin and phenobarbitone by high-performance liquid chromatography

A rapid, sensitive and accurate high-performance liquid chromatographic method for the simultaneous quantitation of phenobarbitone, phenytoin, carbamazepine and carbamazepine-10,11-epoxide in saliva is described. Only small volumes of saliva (100 microliters) are required. Separation of the drugs is achieved by reversed-phase chromatography on a Nova-Pak C18 column, with a mobile phase of acetonitrile-phosphate buffer at a flow-rate of 2.0 ml/min. Detection is effected by ultra-violet absorption at 215 nm. The total run time is under 12.5 min per assay. A precipitation but no extraction step is involved, simplifying the assay method. Salivary concentrations in the range 0.25-25 micrograms/ml for carbamazepine, 0.5-20 micrograms/ml for phenytoin and phenobarbitone and 0.4-20 micrograms/ml for carbamazepine-10,11-epoxide can be measured. Recovery varies from 94 to 108%. The method has been used for routine measurements of anticonvulsants in saliva collected daily from patients with intractable epilepsy.     

Multiple ligand detection and affinity measurement by ultrafiltration and mass spectrometry analysis applied to fragment mixture screening

Binding affinity of a small molecule drug candidate to a therapeutically relevant biomolecular target is regarded the first determinant of the candidate's efficacy. Although the ultrafiltration-LC/MS (UF-LC/MS) assay enables efficient ligand discovery for a specific target from a mixed pool of compounds, most previous analysis allowed for relative affinity ranking of different ligands. Moreover, the reliability of affinity measurement for multiple ligands with UF-LC/MS has hardly been strictly evaluated. In this study, we examined the accuracy of K_d determination through UF-LC/MS by comparison with classical ITC measurement. A single-point K_d calculation method was found to be suitable for affinity measurement of multiple ligands bound to the same target when binding competition is minimized. A second workflow based on analysis of the unbound fraction of compounds was then developed, which simplified sample preparation as well as warranted reliable ligand discovery. The new workflow implemented in a fragment mixture screen afforded rapid and sensitive detection of low-affinity ligands selectively bound to the RNA polymerase NS5B of hepatitis C virus. More importantly, ligand identification and affinity measurement for mixture-based fragment screens by UF-LC/MS were in good accordance with single ligand evaluation by conventional SPR analysis. This new approach is expected to become a valuable addition to the arsenal of high-throughput screening techniques for fragment-based drug discovery.     

Antibody characterization and immunoassays for palytoxin using an SPR biosensor

Palytoxin (PLTX), a polyether marine toxin originally isolated from the zoanthid Palythoa toxica, is one of the most toxic non-protein substances known. Fatal poisonings have been linked to ingestion of PLTX-contaminated seafood, and effects in humans have been associated with dermal and inhalational exposure to PLTX containing organisms and waters. Additionally, PLTX co-occurrence with other well-characterized seafood toxins (e.g., ciguatoxins, saxitoxins, tetrodotoxin) has hindered direct associations of PLTX to seafood-borne illnesses. There are currently no validated methods for the quantitative detection of PLTX(s). As such, a well-characterized, robust, specific analytical technique is needed for the detection of PLTX(s) in source organisms, surrounding waters, and clinical samples. Surface plasmon resonance (SPR) biosensors are ideally suited for antibody characterization and quantitative immunoassay detection. Herein, we describe a newly developed SPR assay for PLTX. An anti-mouse substrate was used to characterize the kinetic values for a previously developed monoclonal anti-PLTX. The characterized antibody was then incorporated into a sensitive, rapid, and selective PLTX assay. Buffer type, flow rate, analyte-binding time, and regeneration conditions were optimized for the antibody–PLTX system. Cross-reactivity to potentially co-occurring seafood toxins was also evaluated. We show that this optimized assay is capable of measuring low- to sub-ng/mL PLTX levels in buffer and two seafood matrices (grouper and clam). Preliminary results indicate that this SPR biosensor assay allows for (1) rapid characterization of antibodies and (2) rapid, sensitive PLTX concentration determination in seafood matrices. Method development information contained herein may be broadly applied to future PLTX detection and/or antibody characterization efforts.     

Label-free detection of protein binding with multisine SPR microchips

Label-free techniques such as surface plasmon resonance (SPR) have used a step-response excitation method to characterize the binding of two biochemical entities. A major drawback of the step response technique is its high susceptibility to thermal drifts and noise which directly determine the minimum detectable binding mass. In this paper we present a new frequency-domain method based on the use of multisine chemical excitation that is much less sensitive to these disturbances. The multisine method was implemented in a PDMS microfluidic chip using a dual channel, dual multiplug chemical signal generator connected to functionalized and reference SPR binding spots. Kinetic constants for the reaction are extracted from the characteristics of the sense spot response versus frequency. The feasibility of the technique was tested using a model system of Carbonic Anhydrase-II analyte and amino-benzenesulfonamide ligand. The experimental signal to noise ratio (SNR) for the multisine measurement is about 32 dB; 7 dB higher than that observed with the single step-response method, while the overall measurement time is twice as long as the step method.     

表面增强拉曼光谱技术在食品检测中的应用研究进展

食品污染是危害公众健康和安全的重要问题,探究灵敏、快速、简单的技术,以便在痕量水平上检测污染物,对保障食品质量安全和风险评价具有十分重要的意义.表面增强拉曼光谱(SERS)是利用光与金、银等纳米结构材料相互作用产生很强的表面等离子激元共振效应,可显著增强吸附在纳米结构表面上分子的拉曼信号,以超灵敏获取样品自身或拉曼探针...     

A review on viral biosensors to detect human pathogens

Rapid identification of viruses has important implications for medical healthcare. Current methods for identification and quantification of particular virus are time consuming and often expensive. Therefore, demand for sensitive and accurate viral biosensors with rapid detection systems is increasing. A hand held biosensing device would give fast, reliable results for identifying and quantitating the number of virus particles in a sample. Techniques currently being applied to achieve this aim include electrochemical biosensors, based on amperometric, potentiometric and impedance measurement, optical biosensors using surface plasmon resonance (SPR), optical fibers and piezoelectric biosensors based on microcantilevers. Future research also looks to the use of nanoparticles and novel nanomaterials as alternate recognition surfaces for use in a variety of sensor formats.     

Monitoring of recombinant survival motor neuron protein using fiber-optic surface plasmon resonance

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. SMA is caused by the homozygous loss of the survival motor neuron 1 (SMN1) gene. A nearly identical copy gene exists known as SMN2, however, due to an aberrant splicing event, the SMN2 gene fails to produce sufficient full-length protein to protect against disease development in the absence of SMN1. While a number of compounds have recently been identified that can stimulate full-length survival motor neuron (SMN) expression from the nearly identical copy SMN2, one of the difficulties has been the lack of a highly reproducible and quantitative means to measure the levels of SMN protein. To develop a technique that allows the rapid and highly sensitive measurement of SMN protein, a Surface Plasmon Resonance (SPR) application has been developed. The ability to quantify unassociated SMN protein and monitor the binding of SMN with other proteins in solution using a SPR sensor in less than 15 min and at low ng mL(-1) levels in HEPES Buffer Saline (HBS) has been achieved. The detection limit for the specific binding of SMN in HBS pH 7.4 solution is 0.99 ng mL(-1) with non-specific binding accounting for approximately 30% of the signal. Quantification of SMN is based on an immunoassay performed on the gold surface of the SPR sensor. 16-mercaptohexadecanoic acid (MHA) was reacted with dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) to form a pre-activated thiol (MHA-NHS). Antibodies for SMN were then coupled to the sensor with the pre-activated thiol. Sensor specificity was examined with mixtures of myoglobin (MG) and SMN. SMN sensor response decreases by more than 60% when MG was added to SMN. The decrease in sensor response can be attributed to non-specific binding of SMN to MG, verified with a sensor for MG.     

Recent progress in the studies of electrochemical interfaces by surface plasmon resonance spectroscopy and microscopy

Surface plasmon resonance (SPR) is a label-free spectroscopic technique that is highly sensitive to various surface reactions. Incorporating SPR into electrochemical measurements has emerged as a powerful method to study both faradaic and non-faradaic processes. SPR microscopy (SPRM) integrates an optical microscope into SPR detection, which further offers high throughput detection and spatially resolved information at an electrode surface and thus, has attracted attention especially in single entity electrochemical studies. In this review, the progress in the studies of electrochemical interfaces by SPR and SPRM during the past two years will be discussed.     

Multiplex suspension array for human anti-carbohydrate antibody profiling

Glycan-binding antibodies form a significant subpopulation of both natural and acquired antibodies and play an important role in various immune processes. They are for example involved in innate immune responses, cancer, autoimmune diseases, and neurological disorders. In the present study, a microsphere-based flow-cytometric immunoassay (suspension array) was applied for multiplexed detection of glycan-binding antibodies in human serum. Several approaches for immobilization of glycoconjugates onto commercially available fluorescent microspheres were compared, and as the result, the design based on coupling of end-biotinylated glycopolymers has been selected. This method requires only minute amounts of glycans, similar to a printed glycan microarray. The resulting glyco-microspheres were used for detection of IgM and IgG antibodies directed against ABO blood group antigens. The possibility of multiplexing this assay was demonstrated with mixtures of microspheres modified with six different ABO related glycans. Multiplexed detection of anti-glycan IgM and IgG correlated well with singleplex assays (Pearson's correlation coefficient r = 0.95-0.99 for sera of different blood groups). The suspension array in singleplex format for A/B trisaccharide, H(di) and Le(x) microspheres corresponded well to the standard ELISA (r > 0.94). Therefore, the described method is promising for rapid, sensitive, and reproducible detection of anti-glycan antibodies in a multiplexed format.     

Headspace gas chromatographic determination of 1,3-butadiene in simulated saliva

A headspace gas chromatographic (GC) method was developed to determine 1,3-butadiene (1,3-BD) in simulated saliva in contact with chewing gum. The calibration graph was linear, and the limit of detection was 0.004 mg/L, which is well below the migration limit for this substance. The headspace GC method provides rapid and reliable analysis for monitoring 1,3-BD migration from chewing gum into simulated saliva. In this paper, we report headspace methodology for sensitive determination of 1,3-BD in chewing gum and results of selected analyses, enabling preliminary assessment of possible exposure to 1,3-BD through migration.     

超高效液相色谱-串联质谱法检测唾液中3种毒品及其代谢物

建立了超高效液相色谱-串联质谱测定唾液中甲基苯丙胺、吗啡、O6-单乙酰吗啡等3种毒品及其代谢物的方法。以乙腈为提取液沉淀蛋白质法提取,采用基质提取溶液配制标准溶液制作定量曲线。采用BEH HILIC超高效液相色谱柱对待测毒品进行分离;采用电喷雾离子源正离子(ESI+)模式和多反应监测(MRM)模式进行质谱分析,以被测毒品的同位素内标进行定量。结果表明,在10、20、50、100 μg/L 4个添加水平下的回收率范围为(68.7±6.5)%～(110.8±4.6)%,日内精密度小于16.5%,日间精密度小于16.3%; 3种毒品的检出限(LOD,以信噪比(S/N)＞3计)和定量限(LOQ,以S/N＞10计)分别为0.02～0.05 μg/L和0.1～0.2 μg/L。方法快速、简便、定量准确、灵敏度高;在1 h之内即可对采集的唾液样品进行毒品定性和定量分析,可用于涉嫌吸毒者的快速认定。     

Discourse on the utilization of polyaniline coatings for surface plasmon resonance sensing of ammonia vapor

Surface plasmon resonance spectroscopy is sensitive to near-surface (<300 nm) chemical and physical events that result in refractive index changes. The non-specific nature of the stimulus implies that chemical selectivity in SPR sensing configurations entirely relies upon the chemical recognition scheme employed. Biosensing applications commonly use surface layers composed of antibodies or enzymes for biomolecular recognition. Monitoring of volatile compounds with SPR spectroscopy, however, has not been widely discussed due to the difficulty in selectively responding to small molecules (<100 Da) in addition to the limited refractive index changes resulting from the interaction between the plasmon wave and volatile compounds.Different strategies explored thus far for sensing of small molecules have relied on optical and electrical changes of the recognition layer upon exposure to the analyte, yielding an indirect measurement. Examples of coatings used for gas-phase sensing with SPR include conducting metal oxides, polymers and organometallic dyes. Electrically conducting polymers, like polyaniline and polypyrrole, display dramatic conductivity changes in the presence of certain compounds. This property has resulted in their routine incorporation into different sensing schemes. However, application of electrically conducting polymers to SPR gas-phase sensing has been limited to a few examples, despite encouraging results.The emeraldine salt form of polyaniline (PAni) demonstrates a decreased electrical conductivity correlated to NH₃ concentration. In this contribution, PAni doped with camphorsulfonic acid (PAni-CSA) was applied to gas-phase sensing of NH₃ by way of SPR spectroscopy. Spectroscopic ellipsometry was used to determine the optical constants (n and k) for emeraldine salt and emeraldine base forms of PAni, confirming the wavelength-dependent response observed via SPR. The analytical performance of the coatings show that a limit of detection of 32 ppm NH₃ based on precision of the mass-flow controllers used and an estimated method limit of detection of ∼0.2 ppm based on three standard deviations of the blank. This is directly comparable to other, more established sensing architectures.