Recent Developments in the Field of Optical Immunosensors Focusing on a Label-Free, White Light Reflectance Spectroscopy-Based Immunosensing Platform.

Optical immunosensors represent a research field of continuously increasing interest due to their unique features, which can mainly be attributed to the high-affinity and specific antibodies they use as biorecognition elements, combined with the advantageous characteristics of the optical transducing systems these sensors employ. The present work describes new developments in the field, focusing on recent bioanalytical applications (2021-2022) of labeled and label-free optical immunosensors. Special attention is paid to a specific immunosensing platform based on White Light Reflectance Spectroscopy, in which our labs have gained specific expertise; this platform is presented in detail so as to include developments, improvements, and bioanalytical applications since the mid-2000s. Perspectives on the field are been briefly discussed as well, highlighting the potential of optical immunosensors to eventually reach the state of a reliable, highly versatile, and widely applicable analytical tool suitable for use at the Point-of-Care.

Comparison of next generation sequencing (NGS) - (SNPs) and capillary electrophoresis (CE) - (STRs) in the genetic analysis of human remains.

A pilot study was performed using two different DNA technology platforms conducted by two laboratories to analyze DNA extracted from 83-year-old, human male skeletal remains from 16 individuals, of which there are no other viable means to identify these war victims. The workflow of the more recent developed ForenSeq Kintelligence Kit and next generation sequencing was compared to that of the standard capillary electrophoresis - short tandem repeat (STR) method (Power Plex ESX17 and Y23 Systems). The findings indicate that greater amount of useful genetic data can be gained with the Kintelligence system across the range of samples under study and particularly for samples in which partial or no STR profiles are obtained. SNP data are more likely to be obtained from degraded samples, like the ones analyzed in this study. Moreover, high volume SNP data are suitable for long distance kinship associations and genetic genealogy databases to develop more investigative leads for future kinship and missing persons cases, a process not feasible by STR typing.

Cortisol Immunosensors: A Literature Review.

Cortisol is a steroid hormone that is involved in a broad range of physiological processes in human/animal organisms. Cortisol levels in biological samples are a valuable biomarker, e.g., of stress and stress-related diseases; thus, cortisol determination in biological fluids, such as serum, saliva and urine, is of great clinical value. Although cortisol analysis can be performed with chromatography-based analytical techniques, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), conventional immunoassays (radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), etc.) are considered the "gold standard" analytical methodology for cortisol, due to their high sensitivity along with a series of practical advantages, such as low-cost instrumentation, an assay protocol that is fast and easy to perform, and high sample throughput. Especially in recent decades, research efforts have focused on the replacement of conventional immunoassays by cortisol immunosensors, which may offer further improvements in the field, such as real-time analysis at the point of care (e.g., continuous cortisol monitoring in sweat through wearable electrochemical sensors). In this review, most of the reported cortisol immunosensors, mainly electrochemical and also optical ones, are presented, focusing on their immunosensing/detection principles. Future prospects are also briefly discussed.

Fast and Accurate Determination of Minute Ochratoxin A Levels in Cereal Flours and Wine with the Label-Free White Light Reflectance Spectroscopy Biosensing Platform.

Ochratoxin A (OTA) is one of the most toxic naturally encountered contaminants and is found in a variety of foods and beverages, including cereals and wine. Driven by the strict regulations regarding the maximum allowable OTA concentration in foodstuff and the necessity for on-site determination, the development of fast and sensitive methods for the OTA determination in cereal flours and wine samples, based on white light reflectance spectroscopy, is presented. The method relied on appropriately engineered silicon chips, on top of which an OTA-protein conjugate was immobilized. A polyclonal antibody against OTA was then employed to detect the analyte in the framework of a competitive immunoassay; followed by the subsequent addition of a biotinylated secondary antibody and streptavidin for signal enhancement. A small size instrument performed all assay steps automatically and the bioreactions were monitored in real time as the software converted the spectral shifts into effective biomolecular adlayer thickness increase. The assay developed had a detection limit of 0.03 ng/mL and a working range up to 200 ng/mL. The assay lasted 25 min (less than 1h, including calibrators/antibody pre-incubation) and was accomplished following a simple sample preparation protocol. The method was applied to corn and wheat flour samples and white and red wines with recovery values ranging from 87.2 to 111%. The simplicity of the overall assay protocol and convenient instrumentation demonstrates the potential of the immunosensor developed for OTA detection at the point of need.

Fast and Sensitive Determination of the Fungicide Carbendazim in Fruit Juices with an Immunosensor Based on White Light Reflectance Spectroscopy.

Carbendazim is a systemic benzimidazole-type fungicide with broad-spectrum activity against fungi that undermine food products safety and quality. Despite its effectiveness, carbendazim constitutes a major environmental pollutant, being hazardous to both humans and animals. Therefore, fast and reliable determination of carbendazim levels in water, soil, and food samples is of high importance for both food industry and public health. Herein, an optical biosensor based on white light reflectance spectroscopy (WLRS) for fast and sensitive determination of carbendazim in fruit juices is presented. The transducer is a Si/SiO2 chip functionalized with a benzimidazole conjugate, and determination is based on a competitive immunoassay format. Thus, for the assay, a mixture of an in-house developed rabbit polyclonal anti-carbendazim antibody with the standards or samples is pumped over the chip, followed by biotinylated secondary antibody and streptavidin. The WLRS platform allows for real-time monitoring of biomolecular interactions carried out onto the Si/SiO2 chip by transforming the shift in the reflected interference spectrum caused by the immunoreaction to effective biomolecular adlayer thickness. The sensor is able to detect 20 ng/mL of carbendazim in fruit juices with high accuracy and precision (intra- and inter-assay CVs ≤ 6.9% and ≤9.4%, respectively) in less than 30 min, applying a simple sample treatment that alleviates any "matrix-effect" on the assay results and a 60 min preincubation step for improving assay sensitivity. Excellent analytical characteristics and short analysis time along with its small size render the proposed WLRS immunosensor ideal for future on-the-spot determination of carbendazim in food and environmental samples.

Development of a Point-of-Care System Based on White Light Reflectance Spectroscopy: Application in CRP Determination.

The development of methods and miniaturized systems for fast and reliable quantitative determinations at the Point-of-Care is a top challenge and priority in diagnostics. In this work, a compact bench-top system, based on White Light Reflectance Spectroscopy, is introduced and evaluated in an application with high clinical interest, namely the determination of C-Reactive protein (CRP) in human blood samples. The system encompassed all the necessary electronic and optical components for the performance of the assay, while the dedicated software provided the sequence and duration of assay steps, the reagents flow rate, the real-time monitoring of sensor response, and data processing to deliver in short time and accurately the CPR concentration in the sample. The CRP assay included two steps, the first comprising the binding of sample CRP onto the chip immobilized capture antibody and the second the reaction of the surface immunosorbed CRP molecules with the detection antibody. The assay duration was 12 min and the dynamic range was from 0.05 to 200 µg/mL, covering both normal values and acute inflammation incidents. There was an excellent agreement between CRP values determined in human plasma samples using the developed device with those received for the same samples by a standard diagnostic laboratory method.

Fast, sensitive and selective determination of herbicide glyphosate in water samples with a White Light Reflectance Spectroscopy immunosensor.

An optical immunosensor based on White Light Reflectance Spectroscopy is described for the determination of the herbicide glyphosate in drinking water samples. The biosensor allows for the label-free real-time monitoring of biomolecular interactions taking place onto a SiO2/Si chip by transforming the shift in the reflected interference spectrum caused by the immunoreaction to effective biomolecular adlayer thickness. Glyphosate determination is accomplished by functionalizing the chip with a protein conjugate of the herbicide followed by a competitive immunoassay format. Prior to the assay, glyphosate derivatization in the calibrators and/or the samples was performed through reaction with succinic anhydride. Under the optimized assay protocol, a detection limit of 10 pg mL-1 was achieved. Recovery values ranging from 90.0 to 110% were determined in spiked bottled and tap water samples, demonstrating the accuracy of the method. In addition, the sensor could be regenerated and re-used for at least 14 times without statistically significant effect on the assay sensitivity and accuracy. The excellent analytical performance and short analysis time (approx. 25 min), combined with the small sensor size, should be helpful for the fast on-site determination of glyphosate in drinking water samples.

Multiplexed mycotoxins determination employing white light reflectance spectroscopy and silicon chips with silicon oxide areas of different thickness.

Biosensing through White Light Reflectance Spectroscopy (WLRS) is based on monitoring the shift of interference spectrum due to the binding reactions occurring on top of a thin SiO2 layer deposited on a silicon chip. Multi-analyte determinations were possible through scanning of a single sensor chip on which multiple bioreactive areas have been created. Nonetheless, the implementation of moving parts increased the instrumentation size and complexity and limited the potential for on-site determinations. Thus, in this work, a new approach, which is based on patterning the sensor surface to create areas with different SiO2 thickness, is developed and evaluated for multi-analyte determinations with the WLRS set-up. The areas of different thickness can be interrogated by a single reflection probe placed on a fixed position over the chip and the reflection spectrum recorded is de-convoluted to the spectra corresponding to each area allowing the simultaneous monitoring of the bioreactions taking place at each one of them. The combination of different areas thickness was optimized using chips with two areas for single analyte assays. The optimum chips were then used for the simultaneous determination of two mycotoxins, aflatoxin B1 and fumonisin B1. A competitive immunoassay format was followed employing immobilization of mycotoxin-protein conjugates onto the SiO2 of different thickness. It was found that the dual-analyte assays had identical analytical characteristics with the respective single-analyte ones. The detection limits achieved were 0.05 ng/mL for aflatoxin B1 and 1.0 ng/mL for fumonisin B1, with dynamic ranges extending up to 5.0 and 50 ng/mL, respectively. The sensor was also evaluated for the determination of the two mycotoxins in whole grain samples (wheat and maize). The extraction protocol was optimized and recoveries ranging from 85 to 115% have been determined. Due to lack of moving parts, the novel multi-analyte format is expected to considerably facilitate the built-up of a portable device for determination of analytes at the point-of-need.

Simultaneous determination of paraquat and atrazine in water samples with a white light reflectance spectroscopy biosensor.

An optical immunosensor based on White Light Reflectance Spectroscopy for the simultaneous determination of the herbicides atrazine and paraquat in drinking water samples is demonstrated. The biosensor allows for the label-free real-time monitoring of biomolecular interactions taking place onto a SiO2/Si chip by transforming the shift in the reflected interference spectrum due to reaction to effective biomolecular layer thickness. Dual-analyte determination is accomplished by functionalizing spatially distinct areas of the chip with protein conjugates of the two herbicides and scanning the surface with an optical reflection probe. A competitive immunoassay format was adopted, followed by reaction with secondary antibodies for signal enhancement. The sensor was highly sensitive with detection limits of 40 and 50 pg/mL for paraquat and atrazine, respectively, and the assay duration was 12 min. Recovery values ranging from 90.0 to 110% were determined for the two pesticides in spiked bottled and tap water samples, demonstrating the sensor accuracy. In addition, the sensor could be regenerated and re-used at least 20 times without significant effect on the assay characteristics. Its excellent analytical performance and short analysis time combined with the small sensor size should be helpful for fast on-site determinations of these analytes.

Development and Bioanalytical Applications of a White Light Reflectance Spectroscopy Label-Free Sensing Platform.

The development of a sensing platform based on white light reflectance spectroscopy (WLRS) is presented. The evolution of the system, from polymer film characterization and sensing of volatile organic compounds to biosensor for the label-free determination of either high (e.g., proteins) or low molecular weight analytes (e.g., pesticides), is described. At the same time, the passage from single to multi-analyte determinations, and from a laboratory prototype set-up to a compact device appropriate for on-site determination, is outlined. The improvements made on both the sensor and the optical set-up, and the concomitant advances in the analytical characteristics and the robustness of the assays performed with the different layouts, are also presented. Finally, the future perspectives of the system, aiming for the creation of a standalone instrument to be used by non-experts, will be discussed.

White light reflectance spectroscopy biosensing system for fast quantitative prostate specific antigen determination in forensic samples.

A label-free biosensor based on white light reflectance spectroscopy for the determination of PSA as semen indicator in forensic samples is presented. The sensor is based on a two-step immunoassay which employs the same polyclonal anti-PSA antibody as capture and detection antibody followed by reaction with streptavidin as a signal enhancement step. The whole assay time was set to 10min; 5min reaction of immobilized antibody with the PSA calibrators or the samples, 3min reaction with the biotinylated anti-PSA antibody and 2min reaction with streptavidin. Following this protocol, a detection limit of 0.5ng/mL was achieved and the assay's linear response range extended up to 500ng/mL. Thus, taking into account the quantification limit of 1.0ng/mL and the average PSA concentration in semen (0.2-5.5mg/mL), semen quantities of a few nanoliters could be detected. The accuracy of the sensor developed was demonstrated through recovery (% recovery ranged from 89.6 to 106) and semen dilution experiments. A linear correlation was found for semen dilutions ranging from 5000 to 360,000. The lack of interference by other bodily fluids was confirmed by analysing stains of blood, urine and saliva prior to and after the addition of semen. Finally, the sensor was evaluated by analysing 51 forensic casework samples which were also analysed with a semi-quantitative membrane strip test (Seratec® PSA), through microscopic detection of spermatozoa, and male DNA identification through detection of Y chromosome. The results obtained with the sensor were in excellent agreement with those provided by an immunoradiometric assay kit (PSA-RIACT) and in complete agreement with the findings using the membrane strip assay, spermatozoa and Y chromosome detection. The excellent analytical performance and small size of the instrument make the sensor developed an attractive tool for use in forensic evidence screening for semen detection.

Simultaneous determination of CRP and D-dimer in human blood plasma samples with White Light Reflectance Spectroscopy.

A dual-analyte assay for the simultaneous determination of C-reactive protein (CRP) and D-dimer in human blood plasma based on a white light interference spectroscopy sensing platform is presented. Measurement is accomplished in real-time by scanning the sensing surface, on which distinct antibody areas have been created, with a reflection probe used both for illumination of the surface and collection of the reflected interference spectrum. The composition of the transducer, the sensing surface chemical activation and biofunctionalization procedures were optimized with respect to signal magnitude and repeatability. The assay format involved direct detection of CRP whereas for D-dimer a two-site immunoassay employing a biotinylated reporter antibody and reaction with streptavidin was selected. The assays were sensitive with detection limits of 25ng/mL for both analytes, precise with intra- and inter-assay CV values ranging from 3.6% to 7.7%, and from 4.8% to 9.5%, respectively, for both assays, and accurate with recovery values ranging from 88.5% to 108% for both analytes. Moreover, the values determined for the two analytes in 35 human plasma samples were in excellent agreement with those received for the same samples by standard diagnostic laboratory instrumentation employing commercial kits. The excellent agreement of the results supported the validity of the proposed system for clinical application for the detection of multiple analytes since it was demonstrated that up to seven antibody areas can be created on the sensing surface and successfully interrogated with the developed optical set-up.