Electrochemical biosensors: towards point-of-care cancer diagnostics

Wide-scale point-of-care diagnostic systems hold great promise for early detection of cancer at a curable stage of the disease. This review discusses the prospects and challenges of electrochemical biosensors for next-generation cancer diagnostics. Electrochemical biosensors have played an important significant role in the transition towards point-of-care diagnostic devices. Such electrical devices are extremely useful for delivering the diagnostic information in a fast, simple, and low cost fashion in connection to compact (hand-held) analyzers. Modern electrochemical bioaffinity sensors, such as DNA- or immunosensors, offer remarkable sensitivity essential for early cancer detection. The coupling of electrochemical devices with nanoscale materials offers a unique multiplexing capability for simultaneous measurements of multiple cancer markers. The attractive properties of electrochemical devices are extremely promising for improving the efficiency of cancer diagnostics and therapy monitoring. With further development and resources, such portable devices are expected to speed up the diagnosis of cancer, making analytical results available at patient bedside or physician office within few minutes.     

Effects of blood sample anticoagulants on lateral flow assays using luminescent photon-upconverting and Eu(III) nanoparticle reporters

Many quantitative and semiquantitative lateral flow (LF) assays have been introduced for clinical analytes such as biomarkers for cancer or acute myocardial infarction (AMI). Various detection technologies involving quantitative analyzing devices have been reported to have sufficient analytical sensitivity and quantification capability for clinical point-of-care tests. Fluorescence-based detection technologies such as quantum dots, Eu(III) nanoparticles, and photon-upconverting nanoparticles (UCNPs) have been introduced as promising solutions for point-of-care devices because of their high detectability by optical sensors. Lateral flow assays can be used for various sample types, e.g., urine, saliva, cerebrospinal fluid, and blood. This study focuses on the properties of serum and plasma because of their relevance in cancer and AMI diagnostics. The limit of detection was compared in LF assays having Eu(III) nanoparticles or UCNPs as reporters and the antibody configurations for two different analytes (prostate-specific antigen and cardiac troponin I (cTnI)). The results indicate a significant effect of anticoagulants in venipuncture tubes. The samples in K₃EDTA tubes resulted in significant interference by decreased reporter particle mobility, and thus the limit of detection was up to eightfold less sensitive compared to serum samples. Despite the matrix interference in the cTnI assay with UCNP reporters, limits of detection of 41 ng/L with serum and 66 ng/L with the Li-heparin sample were obtained.     

Optical Immunosensors for the Efficient Detection of Target Biomolecules

Recently, immunosensors have attracted attention because they are widely applied for the detection of various pathogens. Among the commonly used immunosensors, the optical immunosensor features prominently as an effective tool for the quantification of the amount of antibodies, antigens, or haptens in complex samples with high sensitivity and specificity. However, very few studies provide comprehensive overviews of optical immunosensors. In this review, we present various methods and applications of optical immunosensors in pathogen detection. We introduced a concise definition of optical immunosensors and the principle of using them for detection. We subsequently discuss the main categories of optical immunosensors and their application to the detection of pathogens, as well as their advantages and limitations. Recent publications from 2006 to 2015 on variously designed optical immunosensors have also been updated. We conclude the review with a brief summary and discuss future directions of optical immunosensors.     

Rapid analysis of fatty acid-binding proteins with immunosensors and immunotests for early monitoring of tissue injury

Fatty acid-binding protein (FABP) holds promise for early detection of tissue injury. This small protein (15 kD) appears earlier in the blood than large proteins after cell damage. Combined its characteristics of high concentration tissue contents and low normal plasma values provide the possibility of a rapid rise above the respective reference values, and thus an early indication of the appearance of tissue injury. A general review was presented on the current status of different types of FABP for the detection of tissue injury in patients with myocardial injury, brain injury and also in athletes or horses with skeletal muscle injury.

To take full advantage of the characteristics of the early marker FABP, rapid analysis is a crucial parameter. In this review, an overview of the development of immunoassay for the quantification of FABP in buffer, plasma or whole blood was outlined. The characteristics of different FABP immunosensors and immunotests were described. The feasibility of these immunoassays to be used in routine clinical practice and in emergency case was also discussed.

Nowadays, the improved automated immunoassays (e.g. a microparticle-enhanced turbidimetric immunoassay), less time-consuming bedside immunosensors and immunotests (e.g. a one-step FABP lateral flow immunotest), are the main advance technology in point-of-care testing. With these point-of-care tests, the application of FABP as an early tissue injury marker has a great potential for many clinical purposes.     

Immunosensors for detection of pesticide residues

Immunosensors are biosensors that use antibodies or antigens as the specific sensing element and provide concentration-dependent signals. There is great potential in the applications of immunosensing technologies for rapid detection of pesticide residues in food and environment. This paper presents an overview of various transduction systems, such as electrochemical, optical, piezoelectric, and nanomechanics methods, which have been reported in the literature in the design and fabrication of immunosensors for pesticide detection. Various immobilization protocols used for formation of a biorecognition interface are also discussed. In addition, techniques of regeneration, signal amplification, miniaturization, and antibodies are evaluated for the development and applications of these immunosensors. It can be concluded that despite some limitations of the immunosensing technologies, these immuosensors for pesticide monitoring are becoming more and more relevant in environmental and food analysis.     

Cross-talk-free multiplexed immunoassay using a disposable electrochemiluminescent immunosensor array coupled with a non-array detector

A disposable electrochemiluminescent (ECL) immunosensor array was fabricated on a screen-printed carbon electrode (SPCE) substrate to perform multiplexed immunoassay (MIA) for the first time. The SPCE substrate was composed of an array of four carbon working electrodes, one common Ag/AgCl reference electrode, and one common carbon counter electrode. The immunosensor array was constructed by site-selectively immobilizing multiple antigens on different working electrodes of the SPCE substrate. With a competitive immunoassay format, the immobilized antigens competed with antigens in the sample to capture their corresponding tri(2,2'-bipyridyl)ruthenium(II)-labeled antibodies. The ECL signals from the immunosensors in this array were sequentially detected by a photomultiplier with the aid of a homemade single-pore-four-throw switch. Due to the ECL readout mechanism and the sequential detection mode, it could avoid the cross-talk between the adjacent immunosensors, which was common in other reported immunosensor array. Human, rabbit and mouse immunoglobulin Gs were near-simultaneously assayed as the model analytes. The linear ranges for them were 10-400, 20-400, and 20-400 ng/mL, with detection limits of 2.9, 6.1 and 6.5 ng/mL (S/N=3), respectively. This novel ECL strategy based on immunosensor array coupled with non-array detector provided a simple, sensitive, low-cost and time-saving approach for MIA. It showed great application potential in point-of-care test and field analysis of bio-agents, with mass production potential and high throughput.     

Spatial mapping of cancer tissues by OMICS technologies

Spatial mapping of heterogeneity in gene expression in cancer tissues can improve our understanding of cancers and help in the rapid detection of cancers with high accuracy and reliability. Significant advancements have been made in recent years in OMICS technologies, which possess the strong potential to be applied in the spatial mapping of biopsy tissue samples and their molecular profiling to a single-cell level. The clinical application of OMICS technologies in spatial profiling of cancer tissues is also advancing. The current review presents recent advancements and prospects of applying OMICS technologies to the spatial mapping of various analytes in cancer tissues. We benchmark the current state of the art in the field to advance existing OMICS technologies for high throughput spatial profiling. The factors taken into consideration include spatial resolution, types of biomolecules, number of different biomolecules that can be detected from the same assay, labeled versus label-free approaches, and approximate time required for each assay. Further advancements are still needed for the widespread application of OMICs technologies in performing fast and high throughput spatial mapping of cancer tissues as well as their effective use in research and clinical applications.     

A novel microfluidic immunoassay system based on electrochemical immunosensors: an application for the detection of NT-proBNP in whole blood

Electrochemical immunosensors have attracted great interest in the search for a selective, simple and reliable system for molecular recognition. Presently, electrochemical immunosensors have been widely studied for biomedical molecular's detection, but the regeneration of these immunosensors has restricted their wide application. To prepare a regeneration-free immunosensor, which may be more suitable for clinical determination, a repeatable immunoassay system was developed based on an electrochemical immunosensor with magnetic nanoparticles, biotin-avidin system (BAS) and Fab antibodies for the heart failure markers aminoterminal pro-brain natriuretic peptides (NT-proBNP). At the same time, a microfluidic system was combined into the proposed system, which enabled continuous determination. Using NT-proBNP as a model system, the proposed immunosensor exhibited rapid and sensitive amperometric response to NT-proBNP with good selectivity, stability, and a wide linear range (0.005-1.67 ng/mL and 1.67-4 ng/mL with a detection limit of 0.003 ng/mL under optimal conditions). Importantly, the proposed immunosensor was also suitable for the detection of other proteins and provided new opportunities for disease diagnosis.     

Recent trends in antibody based sensors

This review details recent advances in the fields of immunosensors and closely related immunoassays in the past decade, together with a discussion of possible future trends. Immunosensors can be classified by the way in which they transduce the signal produced upon the formation of an antibody antigen complex. Recent advancements to these methods of detection and transduction are discussed in detail, with particular focus on electrochemical, optical, piezoelectric and magnetic based sensors. The varying applications of these sensors are also discussed. Some of the most significant advances include development of immunosensors for the continuous monitoring of analytes, point of care (PoC) devices, with lower unit costs, automation, reusability and ease of use. Immunosensor technology has advanced at a prolific rate since its conception and has grown into a diverse area of ongoing research.     

Development of immunosensors using carbon nanotubes

With increasing reports on bioterrorism, avian flu, and other bio-threats, rapid and real time detection methods are highly warranted. Studies on developing highly sensitive immunosensors aiming at the early detection and clinical diagnoses of various diseases including cancer are undertaken all over the globe. Carbon nanotubes (CNTs) have been widely discussed as materials with enormous potential for a wide range of in vivo and in vitro bioapplications, ranging from drug delivery to highly sensitive biosensors, owing to their superior electronic and mechanical properties along with nanoscale dimensions. Though a lot of attention has been drawn toward carbon nanotubes for the past 15 years in academia and to a certain extent in industry, CNT-based immunosensors and other applications are still in the nascent stage, and there are many challenges to be overcome for the successful commercialization of the concepts. This article highlights on the recent developments and the possible impacts of carbon nanotube based immunosensors.     

Carbon nanotube electric immunoassay for the detection of swine influenza virus H1N1

A low-cost, label-free, ultra-sensitive electric immunoassay is developed for the detection of swine influenza virus (SIV) H1N1. The assay is based on the excellent electrical properties of single-walled carbon nanotubes (SWCNTs). Antibody-virus complexes influence the conductance of underlying SWCNT thin film, which has been constructed by facile layer-by-layer self-assembly. The basic steps of conventional immunoassay are performed followed by the electric characterization of immunochips at the last stage. The resistance of immunochips tends to increase upon surface adsorption of macromolecules such as poly-L-lysine, anti-SIV antibodies, and SIVs during the assay. The resistance shift after the binding of SIV with anti-SIV antibody is normalized with the resistances of bare devices. The sensor selectivity tests are performed with non-SIVs, showing the normalized resistance shift of 12% as a background. The detection limit of 180 TCID(50)/ml of SIV is obtained suggesting a potential application of this assay as point-of-care detection or monitoring system. This facile CNT-based immunoassay also has the potential to be used as a sensing platform for lab-on-a-chip system.     

Amplification of fluorescence with packed beads to enhance the sensitivity of miniaturized detection in microfluidic chip

This paper reports the pre-concentration of C-reactive protein (CRP) antigen with packed beads in a microfluidic chamber to enhance the sensitivity of the miniaturized fluorescence detection system for portable point-of-care testing devices. Although integrated optical systems in microfluidic chips have been demonstrated by many groups to replace bulky optical systems, the problem of low sensitivity is a hurdle for on-site clinical applications. Hence we integrated the pre-concentration module with miniaturized detection in microfluidic chips (MDMC) to improve analytical sensitivity. Cheap silicon-based photodiodes with optical filter were packaged in PDMS microfluidic chips and beads were packed by a frit structure for pre-concentration. The beads were coated with CRP antibodies to capture antigens and the concentrated antigens were eluted by an acid buffer. The pre-concentration amplified the fluorescence intensity by about 20-fold and the fluorescence signal was linearly proportional to the concentration of antigens. Then the CRP antigen was analyzed by competitive immunoassay with an MDMC. The experimental result demonstrated that the analytical sensitivity was enhanced up to 1.4 nM owing to the higher signal-to-noise ratio. The amplification of fluorescence by pre-concentration of bead-based immunoassay is expected to be one of the methods for portable fluorescence detection system.     

An electrochemical immunosensor for aflatoxin M1 determination in milk using screen-printed electrodes

The production and assembling of disposable electrochemical AFM1 immunosensors, which can combine the high selectivity of immunoanalysis with the ease of the electrochemical probes, has been carried out. Firstly immunoassay parameters such as amounts of antibody and labelled antigen, buffer and pH, length of time and temperature of each steps (precoating, coating, binding and competition steps) were evaluated and optimised in order to set up a spectrophotometric enzyme-linked immunosorbent assay (ELISA) procedure. This assay exhibited a working range between 30 and 160 ppt in a direct competitive format. Then electrochemical immunosensors were fabricated by immobilising the antibodies directly on the surface of screen-printed electrodes (SPEs), and allowing the competition to occur between free AFM1 and that conjugated with peroxidase (HRP) enzyme. The electrochemical technique chosen was the chronoamperometry, performed at -100 mV. Furthermore, studies of interference and matrix effects have been performed to evaluate the suitability of the developed immunosensors for the analysis of aflatoxin M1 directly in milk. Results have shown that using screen-printed electrodes aflatoxin M1 can be measured with a detection limit of 25 ppt and with a working range between 30 and 160 ppt. A comparison between the spectrophotometric and electrochemical procedure showed that a better detection limit and shorter analysis time could be achieved using electrochemical detection.     

System Integration - A Major Step toward Lab on a Chip

Microfluidics holds great promise to revolutionize various areas of biological engineering, such as single cell analysis, environmental monitoring, regenerative medicine, and point-of-care diagnostics. Despite the fact that intensive efforts have been devoted into the field in the past decades, microfluidics has not yet been adopted widely. It is increasingly realized that an effective system integration strategy that is low cost and broadly applicable to various biological engineering situations is required to fully realize the potential of microfluidics. In this article, we review several promising system integration approaches for microfluidics and discuss their advantages, limitations, and applications. Future advancements of these microfluidic strategies will lead toward translational lab-on-a-chip systems for a wide spectrum of biological engineering applications.     

High-sensitivity blood-based detection of breast cancer by multi photon detection diagnostic proteomics

We have developed several new methods for blood-based cancer detection by diagnostic proteomics. Ultrasensitive methods of immunoassay using multiphoton-detection (IA/MPD) increase sensitivity by 200- to 1,000-fold (1 femtogram/mL). This has allowed the measurement of cancer biomarkers with very low concentrations in blood that could not be measured for full patient cohorts with conventional immunoassays. Sensitivity and specificity in cancer detection have been found to be potentiated by use of immunoassay panels which include tissue-specific cancer biomarkers as well as cytokines and angiogenic factors. The ultrasensitive immunoassays revealed that patient to patient variations in the concentrations of individual biomarkers in blood can extend over many orders of magnitude (up to six) and that the distributions of biomarker concentrations over patient cohorts are non-Gaussian. New methods of data analysis which correlate abundances of multiple, different biomarkers have been developed to deal with such data sets. Sensitivity and specificity of about 95% have been achieved for blood-based detection of breast cancer in pilot studies on 250 patients and 95 controls. Pilot studies indicate that this methodology may also allow differentiation of malignant breast cancer from benign lesions and can provide similar sensitivity and specificity for other epithelial cancers such as prostate cancer, ovarian cancer and melanoma. The methods developed for selection, application, and evaluation of very high sensitivity biomarker panels are expected to have general relevance for diagnostic proteomics.     

食品中农药残留的新型生物检测技术研究进展

食品安全是关系国计民生的重大战略问题。农药残留（简称农残）是造成食品质量与安全问题的重要因素。食品中农药残留不仅发生率高，而且超标严重，涉及范围广，长期接触或食用含有农药残留的食品会危害人体健康。传统的农残分析技术已经很难满足多样化食品安全检测的实际需求，现代新型生物技术因特异性强、灵敏度高、简单快速等优势，在食品的农残检测中得到广泛应用和持续发展。分析了食品中农药残留及检测技术现状，综述了酶联免疫吸附测定法、荧光免疫法和生物条形码免疫法等分析技术，及电化学、光学、压电传感器等生物传感技术在食品农残检测中的应用进展，讨论了各种技术的优势和局限，以期为相关研究提供参考，从而进一步提高我国食品农残的检测效率，保障食品安全，促进我国食品行业的可持续发展。     

A comparative study of protein immobilization techniques for optical immunosensors.

This paper presents the results of a study of a number of antibody immobilization techniques for application to optical immunosensors. In particular, well-known methods such as covalent binding and physical adsorption have been extended to the Langmiur-Blodgett method in an attempt to improve the density and possibly the uniformity of orientation of monoclonal antibodies on an optical surface. The surface density of active immobilized antibodies was determined from enzyme immunoassay and their thickness and refractive index were deduced from ellipsometry. It is shown that, although high surface densities (500 ng/cm2) of antibody can be obtained, the major obstacle to the detection of low concentrations of antigens or haptens is the non-specific binding of foreign molecules to the sensing surface.     

Optical immunosensing systems--meeting the market needs

Optical immunosensors and sensing systems are biosensors which produce a quantitative measure of the amount of antibody, antigen or hapten present in a complex sample such as serum or whole blood. The market needs for such devices and their associated instrumentation are reviewed. A brief history of the development of optical immunosensors is presented and the performance of the most well-developed optical immunosensors for meeting these market needs is reviewed. One device, the fluorescent capillary fill device (FCFD) is reviewed in detail with respect to it fulfilling the market needs for an optical immunosensor. Areas for the future development of such sensing systems are also discussed.     

CA 125: the past and the future

Over the last 15 years, substantial progress has been made in understanding the potential and the limitations of the CA 125 assay. More than 2000 papers have been published concerning laboratory and clinical studies of CA 125. The original CA 125 assay utilized the OC 125 antibody that recognizes the CA 125 epitope on a high molecular weight glycoprotein. Despite repeated attempts, the gene encoding the peptide component has not yet been cloned. Monoclonal antibodies have been raised against other epitopes expressed by this molecule, leading to the development of the CA 125-II assay that exhibits less day-to-day variation. Using either assay, elevated levels of CA 125 are detected in a number of benign conditions, including endometriosis. CA 125 is most consistently elevated in epithelial ovarian cancer, but can be expressed in a number of gynecologic (endometrial, fallopian tube) and non-gynecologic (pancreatic, breast, colon and lung) cancers. The best established application of the CA 125 assay is in monitoring ovarian cancer. The rate of decline in CA 125 during primary chemotherapy has been an important independent prognostic factor in several multivariate analyses. Persistent elevation of CA 125 at the time of a second look surgical surveillance procedure predicts residual disease with > 95% specificity. Rising CA 125 values have preceded clinical detection of recurrent disease by at least 3 months in most, but not all studies. Given the modest activity of salvage chemotherapy, this information has not yet impacted on survival. Rising CA 125 during subsequent chemotherapy has been associated with progressive disease in more than 90% of cases. CA 125 may serve as an effective surrogate marker for clinical response in phase II trials of new drugs. CA 125 levels can aid in distinguishing malignant from benign pelvic masses, permitting effective triage of patients for primary surgery. Early detection of ovarian cancer remains the most promising application of CA 125. An algorithm has been developed that estimates the risk of ovarian cancer (ROC) based upon the level and trend of CA 125 values. A major trial has been initiated that uses the ROC algorithm to trigger transvaginal sonography and/or subsequent laparotomy. Such a trial could demonstrate improvement in survival through early detection. This strategy should provide adequate specificity, but sensitivity for early stage disease may not be optimal. In the future, improved sensitivity may be attained using multiple markers and neural network analysis. Most serum tumor markers have been proteins or carbohydrates, but lipid markers such as lysophosphatidic acid deserve evaluation. Genomic and proteonomic technologies should identify additional novel markers.     

Bioseparation and bioanalytical techniques in environmental monitoring

The growing use of antibody-based separation methods has paralleled the expansion of immunochemical detection methods in moving beyond the clinical diagnostic field to applications in environmental monitoring. In recent years high-performance immunoaffinity chromatography, which began as a separation technique in biochemical and clinical research, has been adapted for separating and quantifying environmental pollutants. Bioaffinity offers a selective biological basis for separation that can be incorporated into a modular analytical process for more efficient environmental analysis. The use of immunoaffinity chromatography for separation complements the use of immunoassay for detection. A widely used immunochemical detection method for environmental analyses is enzyme immunoassay. The objective of this paper is to review the status of bioaffinity-based analytical procedures for environmental applications and human exposure assessment studies. Environmental methods based on bioaffinity range from mature immunoassays to emerging techniques such as immunosensors and immunoaffinity chromatography procedures for small molecules.