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.     

Immunosensors

The current trends and future aspects of the research and development of immunosensors are overviewed. A non-labelled immunosensor, whose selectivity depends on immunochemical affinity of an antigen for its corresponding antibody, has been developed as the basis for the potentiometric determination of an antigen, with an antibody-bound membrane or electrode. Non-labelled immunosensors for syphilis antibody, blood typing, human chorionic gonadotropin (HCG), and human serum albumin have been investigated. In contrast with non-labelled immunosensors, labelled immunosensors may be characterized by marked enhancement of sensitivity. Of these labelled immunosensors, enzyme immunosensors that use the chemical amplification of a labelling enzyme for sensitivity are promising. Enzyme immunosensors with an oxygen electrode have been developed to determine AFP, HCG, IgG and toxin. Bioaffinity sensors with a preformed metastable ligand-receptor complex, which are similar to the enzyme immunosensor have been found effective for the determination of thyroxine (T4), biotin, and insulin.     

用于肿瘤标志物检测的电化学免疫传感器

联合检测几种肿瘤标志物,在肿瘤早期诊断中具有重要的临床应用价值。随着纳米技术、流动注射分析技术、微流控技术以及丝网印刷术的迅猛发展,电化学免疫传感器可以在肿瘤标志物的检测中扮演越来越重要的角色。本文主要介绍了电化学免疫传感器的原理及其在肿瘤蛋白标志物检测中的应用情况,并介绍了纳米材料、流动注射分析、微流控等技术在肿瘤标志物免疫传感器中的运用,展望了电化学免疫传感器的前景。     

Advances in ovarian cancer diagnosis: A journey from immunoassays to immunosensors

This review focuses on the technological advancements, challenges and trends in immunoassay technologies for ovarian cancer diagnosis. Emphasis is placed on the principles of the technologies, their merits and limitations and on the evolution from laboratory-based methods to point-of-care devices. While the current market is predominantly associated with clinical immunoassay kits, over the last decade a major thrust in development of immunosensors is evident due to their potential in point-of-care devices. Technological advancements in immunosensors, extending from labeled to label-free detection, with and without mediators, for enhancing proficiencies and reliability have been dealt with in detail. Aspects of the utilisation of nanomaterials and immobilization strategies for enhancing sensitivity and altering the detection range have also been addressed. Finally, we have discussed some distinct characteristics and limitations associated with the recently commericalised technologies used for quantitation of relevant ovarian cancer markers.     

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.     

New amperometric and potentiometric immunosensors based on gold nanoparticles/tris(2,2'-bipyridyl)cobalt(III) multilayer films for hepatitis B surface antigen determinations

Two generic, fast, sensitive and novel electrochemical immunosensors have been developed. Initially, a layer of plasma-polymerized Nafion film (PPF) was deposited on the platinum electrode surface, then positively charged tris(2,2'-bipyridyl)cobalt(III) (Co(bpy)(3)(3+)) and negatively charged gold nanoparticles were assembled on the PPF-modified Pt electrode by layer-by-layer technique. Finally, hepatitis B surface antibody (HBsAb) was electrostatically adsorbed on the gold nanoparticles surface. Electrochemical behavior of the {Au/Co(bpy)(3)(3+)}(n) multilayer film-modified electrodes was studied. Cyclic voltammetry, electrochemical impedance spectroscopy (EIS) were adopted to monitor the regular growth of the multilayer films. The performance and factors influencing the performance of the resulting immunosensors were studied in detail. The multilayer film-modified immunosensor was used for hepatitis B surface antigen (HBsAg) determination via the amperometric and potentiometric immunosensor systems, and both systems provided the same linear ranges from 0.05 to 4.5 microg/mL with different detection limits for the amperometric system 0.005 microg/mL and for the potentiometric system 0.015 microg/mL. The immunosensors were used to analyse HBsAg in human serum samples. Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting HBsAg in the clinical diagnosis. In addition, the multilayer films also showed better stability for 1 month at least.     

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.     

A "signal-on" electrochemical aptasensor for simultaneous detection of two tumor markers

In this paper, we report a "signal-on" electrochemical aptasensor for simultaneous determination of two tumor markers MUC1 and VEGF(165), by using a ferrocene-labeled aptamer-complementary DNA (cDNA) as probe. Since the cDNA immobilized on an electrode surface can hybridize with both MUC1 aptamer and VEGF(165) aptamer to form a long double strand with ferrocene far away from the electrode surface, the probe cannot give electrochemical signal. Nevertheless, the presence of the two tumor markers will inhibit the hybridization of cDNA with the aptamers, thus the distance between ferrocene and the electrode is changed, and a "signal-on" electrochemical method to detect two tumor markers is developed. Experimental results show that the electrochemical signal increases with the addition of either tumor markers, but the biggest electrochemical signal can only be obtained when both tumor markers are present. Therefore, the proposed electrochemical aptasensor can not only detect the two markers but also distinguish their co-existence. It may also display high selectivity and sensitivity towards the detection of the tumor markers, so it might have potential clinical application in the future.     

CEA determination in the follow-up of extracolorectal neoplasms.

CEA was initially described as a tumor and organ specific colorectal antigen, but later found by more sensitive methods in other tumors (stomach, pancreas, lung, breast) and in minor amounts in inflammatory, normal adult and fetal organs of the gastrointestinal tract. The main clinical application of CEA concerns its pretherapeutic and serial determination as circulating antigen in serum and other body fluids by means of CEA-specific, commercially available test kits. By clinical studies a significant correlation has been proven between the pretherapeutic serum CEA level and tumor stages and prognosis. Moreover, serial CEA level changes have been shown a valuable monitor following operation or during radio/chemotherapy anticipating and reflecting the clinical course of disease. In combination with newly established tumor markers, the main clinical indication for CEA determination in addition to colorectal cancer concerns monitoring of patients with stomach (+CA 72-4), lung (+NSE/SCC) and breast cancer (+CA 15-3/MCA).     

Direct determination of urinary pseudouridine by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method was developed for the determination of pseudouridine in urine. This method does not need pretreatment by boronate affinity gel. Therefore, it can be used in screening patients with malignant disease and for monitoring clinical response to chemotherapy with other tumor markers.     

Chiral discrimination using an immunosensor

Based on the stereoselectivity of immunoglobulins, we have developed a new chiral sensor for the detection of low-molecular-weight analytes. Using surface plasmon resonance detection, enantiomers of free, underivatized alpha-amino acids can be monitored in a competitive assay by their interaction with antibodies specific for the chiral center of this class of substances. The sensitivity to the minor enantiomer in nonracemic mixtures exceeds currently available methods; therefore, such immunosensors can readily detect traces of enantiomeric impurities and are attractive for a range of applications in science and industry.     

Molecular imaging of autoimmune diseases and inflammation

Molecular imaging methods allow the noninvasive detection and localization of specific molecules. Agents that report on molecular disease biomarkers can be used to diagnose and monitor disease. Many inflammatory diseases have molecular signatures within altered tissues. Although tissue biopsy is still the gold standard for detecting these signatures, several molecular imaging markers have been developed. Pharmacologic agents that block specific immune molecules have recently entered the clinic, and these drugs have already transformed the way we care for patients with immune-mediated diseases. The use of immunomodulatory drugs is usually guided by clinical assessment of the patient's response. Unfortunately, clinical assessment may miss the signs of inflammation, and many of the serologic markers of immune-mediated diseases correlate poorly with the underlying inflammatory activity within target tissues. Molecular imaging methods have the potential to improve our ability to detect and characterize tissue inflammation. We discuss some of the molecular signatures of immune activation and review molecular imaging methods that have been developed to detect active tissue inflammation.     

BM micrometastases and circulating tumor cells in breast cancer patients: where have we been, where are we now and where does the future lie?

Over the past 15 years early tumor cell dissemination has been detected in patients with breast cancer using sensitive immunocytochemical and molecular assays based on the use of MAb and PCR, respectively. Clinical studies involving more than 4,000 breast cancer patients have now demonstrated that the presence of disseminated tumor cells in BM identified with immuncytochemical assays at primary diagnosis is a strong and independent prognostic factor. The published studies for the detection of disseminated tumor cells in BM fulfill the highest level of evidence as prognostic markers in primary breast cancer. In addition, various assays for the detection of circulating tumor cells in the peripheral blood have been developed recently and some studies suggest a potential clinical relevance of this parameter as a prognostic and predictive factor. Comparative analyzes indicate that the prognostic information derived from BM and blood screening seems to be complementary and not redundant. Advanced methods for molecular characterization of single tumor cells and the surrounding environment have been developed lately, and this approach allows new insights into the metastatic cascade and characterization of targets for therapeutic approaches. Taken together, these findings provide the basis for the implementation of disseminated tumor cells in BM or blood as markers for stratification and assessment of therapies in prospective clinical trials. The valuable information derived from these trials should help to improve future treatment of breast cancer patients.     

New molecular tumor markers for endometrial cancer

Although the International Federation of Gynecology and Obstetrics officially changed the classification system of endometrial cancer from a clinically staged to a surgically staged disease in 1988, optimal management of patients with endometrial cancer is still controversial. Gynecologists happen to experience that patients with tumors that are identical in grade and stage often have significantly different clinical outcomes or responses to therapy. In order to identify an objective biological factor correlating with tumor aggressiveness, many tumor markers have been investigated. So far, CA125 is one of the most reliable tumor marker for adenocarcinoma of the uterus and frequently used in a clinical setting. Recently, with the advent of molecular biological techniques, many genes and regions of the genome related to endometrial cancer have been identified. We undertook a genome-wide screening to detect genetic changes by comparative genomic hybridization (CGH) in primary endometrioid cancers, since CGH analysis provides comprehensive information concerning relative chromosomal losses and gains in tumors by a single hybridization. In this paper, the usefulness of serum tumor markers and the new promising molecular tumor markers for endometrial cancer are discussed.     

Immunoassay of nine serological tumor markers on a hydrogel-based microchip

A prototype test-system for simultaneous quantitative assay of nine tumor markers in blood serum was developed. The main constituent of the test-system is an OM-9 biochip containing immobilized antibodies against nine oncomarkers: α-fetoprotein (AFP), carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG), cancer antigen 15-3 (CA 15-3), cancer antigen 125 (CA 125), cancer antigen 19-9 (CA 19-9), total and free forms of prostate-specific antigen (PSA_tot and PSA_free), and neuron-specific enolase (NSE). The biochip-based two-step sandwich immunoassay procedure for carrying out simultaneous quantitative determination of nine tumor markers in patients’ blood serum was proposed. The main analytical characteristics of the method were obtained. The results suggest that the prototype of the test-system could be a promising instrument for clinical application. The test-system prototype was tested using blood serum samples of oncological patients (252 samples) and healthy donors (185 samples). Increased concentrations of one or more tumor markers above the normal level were found in 76.6% cases of oncological patients and only in 6% cases of healthy donors. For colorectal cancer patients, application of modern statistical methods of data processing in medical research, i.e., receiver operating characteristics analysis (ROC curve) and logistic regression, indicated that the simultaneous assay of nine markers on biochips showed much more diagnostic significance (area under the ROC curve, AUC, was 0.84) than a traditional assay of two tumor markers, CEA and CA 19-9 (AUC = 0.59). The developed biochip-based test-system can be recommended for both the estimation of people’s health, e.g., for standard medical examination, and tracking the tumoral process in the postsurgical period or after specific tumor treatment.     

A disposable two-throughput electrochemical immunosensor chip for simultaneous multianalyte determination of tumor markers

A disposable two-throughput immunosensor array was proposed for simultaneous electrochemical determination of tumor markers. The low-cost immunosensor array was fabricated simply using cellulose acetate membrane to co-immobilize thionine as a mediator and two kinds of antigens on two carbon electrodes of a screen-printed chip, respectively. With two simultaneous competitive immunoreactions the corresponding horseradish peroxidase (HRP) labeled antibodies were captured on the membranes, respectively, on which the immobilized thionine shuttled electrons between HRP and the electrodes for enzymatic reduction of H2O2 to produce detectable signals. The electrochemical and electronic cross-talks between the electrodes could be avoided, which was beneficial to the miniaturization of the array without considering the distance between immunosensors. Under optimal conditions the immunosensor array could be used for fast simultaneous electrochemical detection of CA 19-9 and CA 125 with the limits of detection of 0.2 and 0.4 U/ml, respectively. The serum samples from clinic were assayed with the proposed method and the results were in acceptable agreement with the reference values. The proposed method for preparation of immunosensor array could be conveniently used for fabrication of disposable electrochemical biochip with high throughput and possessed the potential of mass production and commercialization.     

Assessment of tumor vascularization: immunohistochemical and non-invasive methods

Growth of solid tumors beyond a certain mass is dependent on the vascular bed from pre-existing host vasculature. The process of angiogenesis is essential not only for primary tumor growth but also for metastasis. The number of microvessels within the invasive component of a primary tumor reflects the degree of tumor angiogenesis. At present the most widely used method to assess neovascularization is the quantitation of intratumoral microvessel density (IMD) by immunohistochemical methods in which specific markers for endothelial cells are employed. In this paper we analyze the different methods used to assess IMD, as well as their advantages and potential methodological pitfalls. Several studies have shown a close correlation between IMD, tumor growth and the occurrence of metastasis, suggesting that IMD is a prognostic indicator of clinical relevance. Furthermore, preliminary studies suggest that determination of angiogenesis may predict responsiveness to some forms of conventional anticancer therapy. Although the histological microvessel density technique is the current gold standard to characterize tumor angiogenesis, it may not be the ideal tool for clinical purposes because it needs to be performed on biopsy material and does not assess the functional pathways involved in the angiogenic activity of tumors. Non-invasive assessment of tumor vascularity is possible in vivo by means of Doppler sonography, dynamic contrast-enhanced magnetic resonance imaging (MRI) and positron emission tomography (PET). These methods may be preferable to histological assay because they are non-invasive, survey the entire tumor, reflect both anatomic and physiologic characteristics, and may be useful to monitor the activity of antiangiogenic therapies.     

Modeling of immunosensors under nonequilibrium conditions. I. Mathematic modeling of performance characteristics.

Immunosensors for the detection of small analytes that use analyte-enzyme conjugates as signal generators require special attention if operated under nonequilibrium conditions. If the size of the analyte and the analyte-enzyme conjugate differ substantially, the two antigens do not diffuse at the same rate. This can cause time-dependent shifts in the sensitivity of competitive immunoassays. Therefore, immunosensors operating at short incubation times require precise timing that meets closely the specifications for which the sensors were calibrated. As an example, we have analyzed kinetic binding curves for the quantitative determination of progesterone with an immobilized monoclonal antibody and a conjugate between horseradish peroxidase and progesterone as signal generator. Mathematical paradigms have been developed to simulate the diffusion, antigen-antibody complex formation, and competitive binding processes in this analytical system. Dose-response curves obtained under nonequilibrium conditions can vary substantially from those obtained at equilibrium of antigen-antibody interaction. The degree of this variation depends on the performance characteristics of the major components of the immunosensor. The developed mathematical solutions reflect experimental results and can be used to model optimal conditions for immunosensors operating under nonequilibrium conditions. In this paper (Part I), we report on the mathematical modeling of the interaction between analyte, analyte-enzyme conjugate, and an immobilized antibody. In Part II (W. Schramm and S.-H. Paek (1991) Anal. Biochem. 196), we present experimental results and compare them with the theoretical models.     

Carbon nanospheres-promoted electrochemical immunoassay coupled with hollow platinum nanolabels for sensitivity enhancement

Two nanostructures including carbon nanospheres-graphene hybrid nanosheets (CNS-GNS) and hollow platinum nanospheres (HPtNS) were first synthesized by using direct electrolytic reduction and wet chemistry methods, respectively. Thereafter, a specific sandwich-type electrochemical immunoassay was designed for determination of carcinoembryonic antigen (CEA) by using HPtNS-labeled horseradish peroxidase-anti-CEA conjugates (HRP-anti-CEA) as molecular tags and anti-CEA-assembled CNS-GPS as sensing probes. Compared with pure graphene nanosheets, the presence of carbon nanospheres on the graphene increased the surface coverage of the substrate, and enhanced the immobilized amount of primary antibodies. Several labeling protocols, such as HRP-anti-CEA, solid platinum nanoparticle-labeled HRP-anti-CEA, and hollow platinum nanospheres-labeled HRP-anti-CEA, were investigated for determination of CEA and improved analytical features were obtained with hollow platinum nanosphere labeling. With the HPtNS labeling method, the effects of incubation time and pH on the current responses of the immunosensors were also studied. The strong attachment of biomolecules to the CNS-GPS and HPtNS resulted in a good repeatability and intermediate precision down to 10.2%. The dynamic concentration range spanned from 0.001 ng mL(-1) to 100 ng mL(-1) CEA with a detection limit of 1.0 pg mL(-1) at the 3S(blank) level. No significant differences at the 0.05 significance level were encountered in the analysis of 10 clinical serum samples between the developed immunoassay and the commercially available electrochemiluminescent method for determination of CEA.     

Dynamics of tumor hypoxia measured with bioreductive hypoxic cell markers

Hypoxic cells are common in tumors and contribute to malignant progression, distant metastasis and resistance to radiotherapy. It is well known that tumors are heterogeneous with respect to the levels and duration of hypoxia. Several strategies, including high-oxygen-content gas breathing, radiosensitizers and hypoxic cytotoxins, have been developed to overcome hypoxia-mediated radioresistance. However, with these strategies, an increased tumor control rate is often accompanied by more severe side effects. Consequently, development of assays for prediction of tumor response and early monitoring of treatment responses could reduce both over- and undertreatment, thereby avoiding unnecessary side effects. The purpose of this review is to discuss different assays for measurement of hypoxia that can be used to detect changes in oxygen tension. The main focus is on exogenous bioreductive hypoxia markers (2-nitroimidazoles) such as pimonidazole, CCI-103F, EF5 and F-misonidazole. These are specifically reduced and bind to macromolecules in viable hypoxic cells. A number of these bioreductive drugs are approved for clinical use and can be detected with methods ranging from noninvasive PET imaging (low resolution) to microscopic imaging of tumor sections (high resolution). If the latter are stained for multiple markers, hypoxia can be analyzed in relation to different microenvironmental parameters such as vasculature, proliferation and endogenous hypoxia-related markers, for instance HIF1alpha and CA-IX. In addition, temporal and spatial changes in hypoxia can be analyzed by consecutive injection of two different hypoxia markers. Therefore, bioreductive exogenous hypoxia markers are promising as tools for development of predictive assays or as tools for early treatment monitoring and validation of potential endogenous hypoxia markers.