Advances in Optical Detection of Human-Associated Pathogenic Bacteria

Bacterial infection is a global burden that results in numerous hospital visits and deaths annually. The rise of multi-drug resistant bacteria has dramatically increased this burden. Therefore, there is a clinical need to detect and identify bacteria rapidly and accurately in their native state or a culture-free environment. Current diagnostic techniques lack speed and effectiveness in detecting bacteria that are culture-negative, as well as options for in vivo detection. The optical detection of bacteria offers the potential to overcome these obstacles by providing various platforms that can detect bacteria rapidly, with minimum sample preparation, and, in some cases, culture-free directly from patient fluids or even in vivo. These modalities include infrared, Raman, and fluorescence spectroscopy, along with optical coherence tomography, interference, polarization, and laser speckle. However, these techniques are not without their own set of limitations. This review summarizes the strengths and weaknesses of utilizing each of these optical tools for rapid bacteria detection and identification.     

A lab-on-a-chip for spectrophotometric analysis of biological fluids

This paper reports a lab-on-a-chip for application in clinical analysis, especially in the spectrophotometric analysis of biological fluids. It is composed of three parts: (1) a microfluidic system die containing the microchannels fabricated using SU-8 techniques; (2) an optical filtering system based on highly selective Fabry-Perot optical resonators using a stack of CMOS process compatible thin-film layers; (3) a detection and readout system fabricated in a CMOS microelectronic process. The system enables low-cost and selective measurement of the concentration of several biomolecules in biological fluids. Operation is based on optical absorption in a well-defined part of the visible spectrum, defined by the reaction of a specific reagent with a specific biomolecule. Signals proportional to the intensity of the light transmitted through the biological fluid are available at the output in the form of bit streams, which allows simple computer interfacing. Moreover, the optical filtering system enables the measurement using white light illumination, thus avoiding the use of a wavelength dependent light source. This characteristic makes the lab-on-a-chip portable and ensures that the analysis can be performed at any location with instantaneous results, without the use of complex and expensive analysis systems. The quantitative measurement of uric acid and total protein in urine is demonstrated.     

Planar optofluidic chip for single particle detection, manipulation, and analysis

We present a fully planar integrated optofluidic platform that permits single particle detection, manipulation and analysis on a chip. Liquid-core optical waveguides guide both light and fluids in the same volume. They are integrated with fluidic reservoirs and solid-core optical waveguides to define sub-picoliter excitation volumes and collect the optical signal, resulting in fully planar beam geometries. Single fluorescently labeled liposomes are used to demonstrate the capabilities of the optofluidic chip. Liposome motion is controlled electrically, and fluorescence correlation spectroscopy (FCS) is used to determine concentration and dynamic properties such as diffusion coefficient and velocity. This demonstration of fully planar particle analysis on a semiconductor chip may lead to a new class of planar optofluidics-based instruments.     

Reusable split-aptamer-based biosensor for rapid detection of cocaine in serum by using an all-fiber evanescent wave optical biosensing platform

A rapid, facile, and sensitive assay of cocaine in biological fluids is important to prevent illegal abuse of drugs. A two-step structure-switching aptasensor has been developed for cocaine detection based on evanescent wave optical biosensing platform. In the proposed biosensing platform, two tailored aptamer probes were used to construct the molecular structure switching. In the existence of cocaine, two fragments of cocaine aptamer formed a three-way junction quickly, and the fluorophore group of one fragment was effectively quenched by the quencher group of the other one. The tail of the three-way junction hybridized with the cDNA sequences immobilized on the optical fiber biosensor. Fluorescence was excited by evanescent wave, and the fluorescence signal was proportional to cocaine concentration. Cocaine was detected in 450 s (300 s for incubation and 150 s for detection and regeneration) with a limit of detection (LOD) of 165.2 nM. The proposed aptasensor was evaluated in human serum samples, and it exhibited good recovery, precision, and accuracy without complicated sample pretreatments.     

Determination of acyclovir in horse plasma and body fluids by high-performance liquid chromatography combined with fluorescence detection and heated electrospray ionization tandem mass spectrometry

Two methods are presented for the determination of 'respectively' the plasma protein unbound and total concentration of acyclovir in horse plasma and body fluids: first, a liquid-liquid extraction was performed on plasma, combined with HPLC-fluorescence detection for the total plasma concentration; second a more sensitive method using high-performance liquid chromatography combined with heated electrospray ionization tandem mass spectrometry (LC-HESI-MS/MS) was described for plasma and for body fluids analysis. To obtain the unbound concentration of acyclovir in plasma, a simple deproteinization step using a Microcon filter was performed. Ganciclovir was used as an internal standard. Analysis was carried out on an Inertsil 5 ODS-3 column for the HPLC-fluorescence method. For the LC-HESI-MS/MS method a PLRP-S column was used. The limit of quantification (LOQ) for the total concentration was set at 50 and 2 ng mL(-1) for the HPLC-fluorescence method and the LC-HESI-MS/MS method, respectively. The limit of quantification for the unbound concentration was set at 5 ng mL(-1) and at 2 ng mL(-1) for body fluids. The methods were successfully used to perform pharmacokinetic and clinical studies in horses after intravenous and oral dosage of acyclovir and its prodrug valacyclovir.     

Dual-light source excitation for mode-filtered light detection

The sensitivity of a multi-channel mode-filtered light detection system has been enhanced by using a dual-light source irradiation technique. The detection system was constructed from an annular column consisting of a bare optical fibre inserted into a capillary. Sample was introduced through the gap between the fibre and the capillary. A multi-channel charge-coupled device was set on the side of the capillary at which four detection windows could be simultaneously monitored. The changes in the intensities of the mode-filtered light on exposure to various concentrations of ethanol samples from each detection window were monitored. The theoretical studies on the sensitivity of detection of the detection system using dual-light source irradiation have been described. The sensitivity of detection was enhanced when a dual-light source instead of a single-light source was employed. The working concentration range for ethanol was 0-80% (v/v) ethanol. The limit of detection was determined to be 1% (v/v) ethanol. The proposed method has been successfully applied to the determination of ethanol contents of some wine samples. The results were satisfactory compared with values obtained from a standard reference method.     

A data-driven,high-throughput methodology to determine tissue-specific differentially methylated regions able to discriminate body fluids

Tissue-specific differentially methylated regions (tDMRs) are regions of the genome with methylation patterns that modulate gene expression in those tissue types. The detection of tDMRs in forensic evidence can permit the identification of body fluids at trace levels. In this report, we have performed a bioinformatic analysis of an existing array dataset to determine if new tDMRs could be identified for use in body fluid identification from forensic evidence. Once these sites were identified, primers were designed and bisulfite modification was performed. The relative methylation level for each body fluid at a given locus was then determined using qPCR with high-resolution melt analysis (HRM). After screening 127 tDMR's in multiple body fluids, we were able to identify four new markers able to discriminate blood (2 markers), vaginal epithelia (1 marker) and buccal cells (1 marker). One marker for each target body fluid was also tested with pyrosequencing showing results consistent with those obtained by HRM. This work successfully demonstrates the ability of in silico analysis to develop a novel set of tDMRs capable of being differentiated by real time PCR/HRM. The method can rapidly determine the body fluids left at crime scenes, assisting the triers of fact in forensic casework.     

Label-free optical biosensor for detection and quantification of the non-steroidal anti-inflammatory drug diclofenac in milk without any sample pretreatment

A label-free optical biosensor for detection and quantification of diclofenac in bovine milk has been developed. This was achieved by using reflectometric interference spectroscopy as detection method. In a first step, the immunosensor was developed and optimised in buffer concerning sensitivity, selectivity, stability and reproducibility. By comparing recovery rates—not only the good intra- but also the good inter-chip—reproducibility could be proven. Consequently, the assay was transferred in the more complex matrix milk. By utilising an optimised surface modification and evaluation method, matrix effects could successfully be prevented or circumvented. As a result, the developed immunosensor does not need sample pretreatment at all. By obtaining a limit of detection of 0.112 μg L^?1 (0.108 μg kg^?1), the capability of the developed biosensor is comparable or better than those of standard detection methods. Moreover, the presented biosensor reaches the range of the maximum residue limit (0.1 μg kg^?1) set by the European Union. Thus, for the first time, diclofenac was successfully quantified at relevant levels in milk by using an optical biosensor.     

Enzyme-catalyzed amperometric oxidation of neurotransmitters in chip-capillary electrophoresis

The determination of biogenic monoamines by enzyme-catalyzed oxidation after electrophoretical separation on a microfluidic chip decreases their detection limits significantly. An amperometric system with a chemically amplified response for neurotransmitters and their metabolites is presented. The principle is the rapid cyclic oxidation of the analyte on the amperometric detector in the presence of the redoxactive enzyme glucose oxidase in the capillary electrophoresis buffer. With this approach, detection limits in the range of 10(-7)-10(-8) M could be reached. Because of the good linearity between the current response and the concentration of catecholamines and their metabolites at concentrations up to 300 microM, this method is attractive for the analytical detection at low concentration levels such as in biological fluids.     

Electrophoresis with intermittent scanning of the migration path: detection of resolution within shortened time.

Intermittent optical scanning (by detection of optical density or fluorescence) of the electrophoretic migration path was applied to the resolution of two dyes under an arbitrary set of conditions. Scanning at 5-min intervals allows for detection of resolution between the two zones at least 3 times faster than conventional automatic zone detection employing a detector at the end of the migration path. This result promises that replacing stationary by mobile detectors in general would result in a substantial time saving for automated detection of electrophoretic zones.     

Ultrasensitive absorption spectroscopy of optically-trapped aerosol droplets

High-sensitivity optical absorption measurements on individual sub-picoliter aqueous droplets are reported using aerosol optical tweezers to simultaneously manipulate and characterize a sample droplet and a control droplet for comparison. It is demonstrated that the detection sensitivity to trace analytes is set by the weak absorption by the solvent, water, and that absorbances less than 5 x 10(-7) can be measured over pathlengths of less than 10 microm. The potential applications of this approach to analyze aerosol particle composition and to perform trace analysis are discussed.     

Development of an integrated capillary electrophoresis/sensor for L-ascorbic acid detection

A CE/biosensor for measuring ascorbic acid was developed by coupling a polyaniline optical sensor and capillary electrophoresis (CE). The capillary column was partially modified with a thin film of polyaniline redox sensitive material. Ascorbic acid was detected by monitoring the changes in optical absorbance occurring to the polyaniline film upon the reduction reaction. The sensor response (change in optical absorbance at 650 nm) is proportional to the concentration of ascorbic acid over a range of 2.5-250 mg/L and the response range has shown a clear dependence on the characteristics of the polymerized film. High specificity and sensitivity of the present method, low sample consumption, short times of response (ca. 2 min) and the reproducibility of the results demonstrate that the CE/polyaniline-sensor could be further employed in the study of the relation between the content of L-ascorbic acid in body fluids and clinical parameters, e.g., cell ageing.     

Selectively detecting attomolar concentrations of proteins using gold lined nanopores in a nanopore blockade sensor

Disease diagnosis at earlier stages requires the development of ultrasensitive biosensors for detecting low-abundance biomarkers in complex biological fluids within a reasonable time frame. Here, we demonstrate the development of an ultrasensitive nanopore blockade biosensor that can rapidly diagnose a model protein biomarker, prostate-specific antigen (PSA) with high selectivity. The solid-state nanopores have gold located only along the length of the nanopore whilst the rest of the membrane is silicon nitride. The orthogonal use of materials allows nanopore arrays with a different surface chemistry inside the nanopore relative to the rest of the membrane to be fabricated. The importance of this differential surface chemistry is it can improve the detection limit of nanopore blockade sensors in quantitative analysis. Based on such functionalized nanopore devices, nanopore blockade sensors lower the limit of detection by an order of magnitude and enable ultrasensitive detection of PSA as low as 80 aM. The findings from this study open new opportunities for nanopore sensors in further developments including optical detection and ultralow detection limit biosensing at complex biological fluids.

Selective detection of attomolar proteins was achieved using gold lined nanopores in a nanopore blockade sensor.     

Mass spectrometry detection as an innovative and advantageous tool in ligand exchange capillary electrophoresis

The copper(II) complex of a modified cyclodextrin, namely 6-mono-deoxy-6-[4-(2-aminoethyl)imidazolyl]-β-CD (CDmh), previously synthesized and characterized in our laboratory and already used as chiral selector for ligand exchange capillary electrophoresis (LECE) with optical detection, is investigated here in LECE using electrospray-mass spectrometry (ESI-MS) as the detection device. The potential of this hyphenated method is compared with the results previously obtained with optical detection by studying the chiral resolution of tryptophan racemate. Chiral separation conditions compatible with LECE-ESI-MS could be achieved based on the figures of merit obtained by LECE-UV. Interestingly, the values of LOD obtained by LECE-ESI-MS were significantly better than those obtained by LECE-UV and thus, ESI-MS detection seems to open new perspectives in chiral separations by LECE.     

Development of multianalyte sensor arrays composed of chemically derivatized polymeric microspheres localized in micromachined cavities.

The development of a chip-based sensor array composed of individually addressable polystyrene-poly(ethylene glycol) and agarose microspheres has been demonstrated. The microspheres are selectively arranged in micromachined cavities localized on silicon wafers. These cavities are created with an anisotropic etch and serve as miniaturized reaction vessels and analysis chambers. A single drop of fluid provides sufficient analysis media to complete approximately 100 assays in these microetch pits. The cavities possess pyramidal pit shapes with trans-wafer openings that allows for both fluid flow through the microreactors/analysis chambers and optical access to the chemically sensitive microspheres. Identification and quantitation of analytes occurs via colorimetric and fluorescence changes to receptor and indicator molecules that are covalently attached to termination sites on the polymeric microspheres. Spectral data are extracted from the array efficiently using a charge-coupled device allowing for the near-real-time digital analysis of complex fluids. The power and utility of this new microbead array detection methodology is demonstrated here for the analysis of complex fluids containing a variety of important classes of analytes including acids, bases, metal cations, metabolic cofactors, and antibody reagents.     

Characterisation and optimisation of an immunoprobe for triazines

The characterisation and optimisation of an optical immunoassay with label free detection based on Reflectometric Interference Spectroscopy (RIfS) is presented. The immunoprobe is operated in a sequential scheme, where Fab-fragments react with analyte molecules in a first step. In a second step the optical transducer is used to quantify the amount of unoccupied Fab- fragments in the reaction mixture binding to the hapten-modified transducer surface. For optimisation of the test, the Fab-fragment concentration was varied between 2x10(-8) mol/l and 2.5x 10(-9) mol/l. Down to a concentration of 5x10(-9) mol/l a reduction in the limit of detection has been observed. At the lowest concentration investigated no further improvement has been found due to a reduced binding of the analyte and a strong decrease of antibody binding at the transducer surface. This finding could be explained by the thermodynamics of the antigen-antibody reaction and the performance of the optical transducer used. The limit of detection obtained is discussed with respect to thermodynamics, transducer characteristics and immunoprobe test format.     

A simple kinetic model of antigen-antibody reactions in particle-enhanced light scattering immunoassays

Polymer colloids coated by antibodies are used in diagnostic tests for the detection of antigens in biological fluids. We present in this paper a simple kinetic model for the optical monitoring of the formation of specific complexes between antigen and antibody with amplification by latex beads. The antibodies are physical or chemically adsorbed onto sulphonate polystyrene or chloromethylstyrene particles respectively. This is a very simple model combining the La Mer idea of optimum surface coverage with Langmuir adsorption of antigen molecules. The kinetic model explains reasonably well the optical response of immunolatex prepared by both methods of immobilization of antibodies on polymer carriers. According to the results obtained with this model the percentage of active IgG on latex particles is extremely low, with a maximum of a 5%.     

手性试剂柱前衍生化高效液相色谱法测定α-苯乙胺的光学纯度

建立了一种简便的手性试剂柱前衍生化反相高效液相色谱测定α-苯乙胺光学纯度的方法。采用2,3,4,6-四-O-β-D-吡喃葡萄糖异氰酸酯(GITC)对α-苯乙胺进行衍生化,优化了衍生化反应参数;使用Agilent Zorbax C18色谱柱分离衍生化产物,流动相为甲醇-磷酸盐缓冲溶液(pH 3.0)(体积比为58:42),流速1.0 mL/min,检测波长241 nm,柱温为30 ℃。实验结果表明,α-苯乙胺两个对映体的衍生化产物分离良好,在0.15～15.0 mg/L范围内呈现良好的线性关系。方法的检出限为0.05 mg/L,定量限为0.15 mg/L,日内和日间精密度考察中测定值的相对标准偏差(RSD)均小于0.5%。建立的方法适用于α-苯乙胺的质量控制。     

A simple,low-cost,remote fiber-optic micro volume fluorescence flowcell for capillary flow-injection analysis

A small volume flowcell for fluorescence detection in capillary flow injection (CFI) analysis has been created by using a low cost, commercially available fluidic device. Fluorescence detection is achieved using an optical fiber to deliver excitation light to the sample flowing through the device and another optical fiber to collect fluorescence emission. The flowcell is a standard fluidic cross with a swept volume of 721 nL. Optical fibers were oriented at right angles using standard sleeves and ferrules to set their position near the cross intersection. Multiple excitation sources were used including a low power UV laser and blue and UV light emitting diodes (LED). The full emission spectrum detection limits, using the laser, for fluorescein and bovine serum albumin (BSA) were 0.30 ppb and 2.1 x 10(-4)% (w/w), respectively. Two fluidic crosses were used in series for multi-wavelength fluorescence excitation using fiber-optically coupled LED.     

Detection of oxilofrine in plasma and urine by high-performance liquid chromatography with electrochemical detection and comparison with gas chromatography-mass spectrometry

A high-performance liquid chromatographic (HPLC) method with electrochemical detection for the determination of oxilofrine [1-(4-hydroxyphenyl)-2-methylaminopropanol] in human plasma and urine (before and after cleavage of the metabolic conjugates) is described. Isolation from biological fluids is performed batchwise by weak acid cation exchange. Separation of plasma and urine components is achieved on a reversed-phase C18 column as an ion pair with heptanesulphonic acid. For amperometric detection the potential of the electrode was set at 0.95 V versus an Ag/AgCl reference electrode. The detection limit for oxilofrine in plasma is 1 ng/ml and in urine 12.5 ng/ml at a signal-to-noise ratio of 2.0 using 1.0 ml of plasma and 0.02 ml of urine. The method was compared with a gas chromatographic-mass spectrometric method and showed a good concordance for plasma (r = 0.996) and urine (r = 0.994). With the HPLC method it is also possible to determine related sympathomimetic drugs, e.g., etilefrine, norefenefrine or octopamine, after a slight modification of the mobile phase.