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Editorial

Overconsumption of biotin (5–100 mg daily) as a supplement by the general population poses a significant problem for clinical immunoassays (IAs) based on biotin–streptavidin (SA) interactions. This affinity pair has been exploited in immunoassays because of its avidity, sensitivity, specificity, and stability. The elevated biotin level in plasma varies from patient to patient, and its severe interference cannot easily be predicted and quantified. Thus, immunoassay manufacturers must investigate the biotin interference in the developed immunoassays to satisfy the threshold of 3510 ng/mL (14 367 nM), as stipulated by the FDA. There is no concrete solution to circumvent the biotin interference without extra costs and technical difficulties, albeit different strategies have been attempted. They include the IA format with biotinylated reagents prebound to streptavidin, the removal of biotin from the specimen, sample treatment, and biotin interference-free assays. The general public has been instructed to stop taking biotin supplements for 48 h or even weeks before the test, depending on the specific test, dose, and frequency of biotin uptake. As lab-based techniques cannot accommodate an enormous number of public samples, a rapid analytical procedure for biotin is urgently needed to quantify for its interference in immunoassays.

Rapid detection of human coronavirus disease 2019, termed as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or COVID-19 infection, is urgently needed for containment strategy owing to its unprecedented spreading. Novel biosensors can be deployed in remote clinical settings without central facilities for infection screening. Electrochemical biosensors serve as analytical tools for rapid detection of viral structure proteins, mainly spike (S) and nucleocapsid (N) proteins, human immune responses, reactive oxygen species, viral ribonucleic acid, polymerase chain reaction by-products, and other potential biomarkers. The development of point-of-care testing devices is challenging due to the requirement of extensive validation, a time-consuming and expensive step. Together with specific biorecognition molecules, nanomaterial-based biosensors have emerged for the fast detection of early viral infections.

There have been tremendous advances in in vitro diagnostics (IVD) for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the confirmatory clinical diagnosis is made by real-time reverse transcriptase polymerase chain reaction (RT-PCR), lateral flow immunoassay (LFIA) based viral antigen (Ag) detection is used for mass population screening at point-of-care (POC) settings. The rapid RT-PCR tests (such as from Cepheid and Bosch) have an assay duration of less than 40 min, while most rapid Ag tests (such as Abbott’s BinaxNOW™ COVID-19 Ag card) have an assay duration of about 15 min. Of interest is the POC molecular test (ID NOW™) from Abbott that takes less than13 min. Similarly, many immunoassays (IAs), i.e., automated chemiluminescent IA (CLIA), manual ELISA, and LFIA, have been developed to detect immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A (IgA) produced in subjects after SARS-CoV-2 infection. Many IVD tests have been approved by the United States Food and Drug Administration (FDA) under emergency use authorization (EUA), and almost all IVD tests are Conformité Européenne (CE) certified.

Graphene, a relatively new two-dimensional (2D) nanomaterial, possesses unique structure (e.g. lighter, harder, and more flexible than steel) and tunable physicochemical (e.g. electronical, optical) properties with potentially wide eco-friendly and cost-effective usage in biosensing. Furthermore, graphene-related nanomaterials (e.g. graphene oxide, doped graphene, carbon nanotubes) have inculcated tremendous interest among scientists and industrials for the development of innovative biosensing platforms, such as arrays, sequencers and other nanooptical/biophotonic sensing systems (e.g. FET, FRET, CRET, GERS). Indeed, combinatorial functionalization approaches are constantly improving the overall properties of graphene, such as its sensitivity, stability, specificity, selectivity, and response for potential bioanalytical applications. These include real-time multiplex detection, tracking, qualitative, and quantitative characterization of molecules (i.e. analytes [H2O2, urea, nitrite, ATP or NADH]; ions [Hg2+, Pb2+, or Cu2+]; biomolecules (DNA, iRNA, peptides, proteins, vitamins or glucose; disease biomarkers such as genetic alterations in BRCA1, p53) and cells (cancer cells, stem cells, bacteria, or viruses). However, there is still a paucity of comparative reports that critically evaluate the relative toxicity of carbon nanoallotropes in humans. This manuscript comprehensively reviews the biosensing applications of graphene and its derivatives (i.e. GO and rGO). Prospects and challenges are also introduced.

A sensitive and rapid sandwich immunoassay (IA) was developed for human lipocalin-2 (LCN2) by functionalizing a KOH-treated polystyrene microtiter plate with multiwalled carbon nanotubes (MWCNTs) dispersed in 3-aminoproyltriethoxysilane (APTES). The significantly increased surface area due to the presence of MWCNTs led to a high immobilization density of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) activated anti-LCN2 capture antibodies (Ab). The anti-LCN2 Ab-bound MTPs were stable for 6 weeks when stored in 0.1 M PBS, pH 7.4 at 4 °C. The IA detects LCN2 from 0.6 to 5120 pg mL−1 with a limit of detection (LOD) and limit of quantification (LOQ) of 0.9 pg mL−1 and 6 pg mL−1, respectively. The assay offered a ~50-fold lower LOD and ~3-fold faster IA, compared to a commercial sandwich enzyme-linked immunosorbent assay kit.

Human fetuin A (HFA) plays a prominent pathophysiological role in numerous diseases and pathophysiological conditions with considerable biomedical significance; one example is the formation of calciprotein particles in osteoporosis and impaired calcium metabolisms. With impressive advances in in vitro diagnostic assays during the last decade, ELISAs have become a workhorse in routine clinical diagnostics. Recent diagnostic formats involve high-sensitivity immunoassay procedures, surface plasmon resonance, rapid immunoassay chemistries, signal enhancement, and smartphone detection. The current trend is toward fully integrated lab-on-chip platforms with smartphone readouts, enabling health-care practitioners and even patients to monitor pathological changes in biomarker levels. This review provides a critical analysis of advances made in HFA assays along with the challenges and future prospects.

Immunoassays play a prominent role in the food industry for the detection of single or multiple analytes in food samples, thereby contributing to the development of high-quality food products. There is a need for stringent bioanalytical testing of food samples and robust statistical analysis taking into account the growing concerns for public health and safety. Statistical analysis plays a prominent role in the food industry for the determination of various bioanalytical parameters, which should comply with the recent guidelines provided by the regulatory authorities. The guidelines should be updated regularly based on the continuously evolving technologies and recent trends in food testing. The developed immunoassay should be precise, accurate, highly sensitive, specific, and robust for intended end-user application, i.e., the detection of analytes in food products. This chapter provides an overview of the critical bioanalytical parameters and the importance of statistical analysis in immunoassays for food analytes.

Carbon nanotubes, graphenes, and their hybridized composites with nanoparticles have been attempted to establish a direct electrical communication between the recognition biomolecule and its underlying electrode surface. This review (with 133 refs.) focuses on advances, strategies and technical challenges in the development of reagentless electrochemical biosensors for glucose with enhanced detection sensitivity, selectivity, and simplicity. Specifically, the review commences with a discussion of the relevance of direct electron transfer (DET) in biosensing together with the fundamental of electro-enzymology and kinetics. General aspects of glucose oxidase (GOx), the most popular enzyme with a flavin cofactor, are discussed in view of its historical and important role in the development of electrical biosensors for blood glucose. The next section assesses DET of GOx based on the Marcus theory and the Laviron formalism. The reorganizational energy of the Marcus model and the overpotential play an important role in reaction kinetics and affect the rate of electron transfer significantly. The presence of nanomaterials, particularly for graphene oxide, decreases the electron transfer distance between the enzyme redox center and the underlying electrode surface well beyond 15 A. The improper Marcus-Hush-Chidsey integral is now simplified to estimate the rate of electron transfer with very good accuracy. Critiques, technical challenges, and future possibilities of glucose electrodes with respect to DET are also presented and discussed.

A surface plasmon resonance (SPR) biosensor has been developed for rapid immunoassay of procalcitonin (PCT) with high detection sensitivity and reproducibility. The 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)-activated protein A (PrA), diluted in 1% (v/v) 3-aminopropyltriethoxysilane (APTES) was dispensed on a KOH-treated Au-coated SPR chip, resulting in the covalent binding of PrA in 30 min. This “single-step” PrA immobilization strategy led to the oriented binding of the anti-PCT antibody (Ab) on a PrA-functionalized gold (Au) chip. The leach-proof immobilization procedure is five-fold faster than conventional counterparts, enabling high detection specificity and reproducibility. The IA detects 4–324 ng mL −1 of PCT with a limit of detection (LOD) and a limit of quantification (LOQ) of 4.2 ng mL −1 and 9.2 ng mL −1 , respectively. It was capable of detecting PCT in real sample matrices and patient samples with high precision. The Ab-bound SPR chips were stable for more than five weeks.

Point-of-care (POC) technologies have advanced considerably during the last decade to pave the way to the emergence of next-generation healthcare monitoring and management. The most prominent developments encompass the smartphone (SP)-based technologies, paper-based assays (PBA), lab-on-a-chip (LOC) platforms, microfluidic operations, new biosensors, rapid assay formats, automated and fully integrated assay technologies, prolonged reagent storage concepts, and novel bioanalytical technologies. The advances in complementary technologies would provide considerable support to the development of critically improved POC technologies. Although paper- and LOC-based assays are cost-effective and simple, emerging SP-based technologies become the ideal POC solution for healthcare due to their enormous outreach and enriched features. Such POC technologies could increase the outreach of healthcare for remote and decentralized settings worldwide. The recent trend is strongly inclined toward mobile healthcare (mH), which would lead to critically improved healthcare monitoring and management.

Immunoassays (IAs) play a prominent role in in vitro diagnostics (IVD) of single or multiple analytes in patient samples, a prerequisite for the monitoring and management of health. Stringent bioanalytical testing in parallel with robust statistical analysis stems from the growing concerns for public health and safety. In addition, the prominent role of statistical analysis should adhere to the recent guidelines provided by the regulatory authorities. The guidelines are regularly updated as reflected by continuously evolving technologies and recent trends in IVD and point-of-care testing (POCT). The IA must provide precision, accuracy, high sensitivity, specificity, reproducibility, and robustness. This chapter provides an overview of the critical bioanalytical parameters and their determination, as desired by the regulatory guidelines.

Smartphone-based personalized mobile healthcare devices (SPMHDs) have become efficient with cost-effectiveness for monitoring and management of healthcare, particularly at remote, decentralized, and personal settings. The last few years have witnessed a surge in commercial SPMHDs for tracking blood pressure, physical activity, blood glucose, body weight, body analysis, pulse rate, electrocardiogram, blood oxygen saturation, and sleep quality. As equipped with advanced Bluetooth technology, Cloud computing, smart application, and telemedicine capabilities, SPMHDs are capable of real-time “on-site” analysis and increasing the user’s compliance by providing constant alerts and notifications. Moreover, they have the most extensive outreach as smartphones have become ubiquitous. With continuous innovation and improvement in mobile healthcare (mH), the next-generation SPMHDs will play a critical role in personalized healthcare to reduce the healthcare costs with improved health outcomes. This chapter provides a comprehensive overview, prospects, and applications of the commercial SPMHDs along with the challenges.

The rapid advances in point-of-care testing (POCT), mobile healthcare (mH), and smart applications are paving the way toward better healthcare monitoring and management of chronic diseases. In the "not too distant" future, many if not most of the routine tests for chronic diseases could be simply performed by the patients in their homes, offices, and custom settings. The test results are then transmitted securely to the certified healthcare professionals, probably via the Cloud, and stored in the patients’ electronic health record (EHR). The patients and their doctors could see the latest results and trend in the test results, enabling them to take timely decisions and perform the desired intervention for better healthcare management. The interface of mH devices to smartphones (SPs), smartwatches, and other gadgets would further improve the compliance by patients as the care provider could set up customized text alerts and alarm for the tests, medication, and physical/lifestyle/medical intervention. This chapter offers a view of the future trends for next-generation personalized and integrated healthcare for chronic diseases.

Paper-based point-of-care (POC) immunoassays (IA) enable the detection of analytes at the remote, decentralized, and personalized settings. Based on their low-cost, simplicity, and rapid analyte detection, they are ideal for POC diagnostic applications in developing countries, which have limited healthcare resources, personnel, and infrastructure. They obviate the limitations of conventional immunodiagnostic assays and the upcoming automated immunoassays, such as the need for highly-skilled analysts, costly infrastructure, bulky and expensive instruments, continuous power supply, and complex process steps. The emerging trend is toward the development of fully-integrated paper-based IAs (PIAs) that can be read by smartphones (SP) or smart readers. This chapter discusses the various PIAs that have been developed to date together with the future trends and challenges.

Smartphone (SP)-based devices and associated tools have emerged as ideal next-generation point-of-care devices (POCD) for in vitro diagnostics (IVD) of important physiological parameters including blood glucose level. Such devices with advanced features are anticipated to play an enhanced role in the future of cost-effective mobile healthcare (mH) and personalized medicine. The detection principle of the diversified SP-IVD devices encompasses optical, surface plasmon resonance, lateral flow, or electrochemical methods. Another appealing approach is the transformation of SP into a compact and inexpensive microscope or cytometer to detect biomolecules, metabolites, biomarkers, and pathogens. The exponential development of SP-IVD technologies will foster their widespread use in personalized mH in remote settings and, particularly, in developing countries with limited healthcare resources. The extent of private health data created by SP-IVD will herald the enlargement of Cloud-based technologies to securely transmit, store, and retrieve such confidential data across medical fraternity and policymakers.

The point-of-care (POC) diabetes management has witnessed significant improvement during the recent years. The current generation of diabetes management softwares has many striking features such as simple analysis, better data visualization, prediction of the trend, and data safety. Most diabetes management softwares have been developed by the topmost in vitro diagnostic (IVD) companies, such as Abbott, Roche, Dexcom, Medtronic, LifeScan, etc., which account for the predominant market share of POC diabetic blood glucose monitoring market. However, during the last decade, several new companies, such as iHealth, have also developed innovative POC diabetic devices and smart applications. Cellphones (CPs) have emerged as the ideal POC device for personalized mobile healthcare. The number of CP users has already crossed 7 billion, which accounts for 98% of the world population. CP-based blood glucose monitoring (BGM) devices and healthcare applications have further led to the development of prospective smart applications for diabetic glucose monitoring and management. The emergence of Cloud computing and wearable devices, such as smart watches, emphasize the critical role of smart applications in providing improved mobile health and telemedicine tools for diabetic management.

Considerable advances in point-of-care testing (POCT) devices stem from innovations in cellphone (CP)-based technologies, paper-based assays (PBAs), lab-on-a-chip (LOC) platforms, novel assay formats, and strategies for long-term reagent storage. Various commercial CP platforms have emerged to provide cost-effective mobile health care and personalized medicine. Such assay formats, as well as low-cost PBAs and LOC-based assays, are paving the way to robust, automated, simplified, and cost-effective POCT. Strategies have also been devised to stabilize reagent storage and usage at ambient temperature. Nevertheless, successful commercialization and widespread implementation of such clinically viable technologies remain subject to several challenges and pending issues.

The last decade has witnessed tremendous advances in the use of graphene and graphene-based nanocomposites for the fabrication of electrochemical (EC) sensors and biosensors with improved analytical performance. With unique and highly desirable morphology, chemical/thermal stability and EC properties, the graphene-based materials are paving way to the implementation of mediatorless EC detection schemes with direct electron transfer. This approach enables the development of highly performed biosensors with respect to detection sensitivity, precision, specificity, and stability. This review provides a comprehensive overview of the field apart from providing intensive information of the fabrication, properties, characterization and EC applications of graphene and its nanocomposites. Two key challenges, the lack of international regulatory guidelines for nanotoxicity analysis and potential mass production of analytical devices, will also be discussed along with the trends in nanobiotechnology and the requirements in healthcare and industrial applications.

This chapter systematically presents the non-invasive POCT methods currently available to date. Furthermore, it explains why long-demanded non-invasive analysis nevertheless rarely provides clinically evaluable results.

Smartphone (SP)-based bioanalytical devices and platforms have been employed for immunoassays to achieve superior bioanalytical performance, low cost, rapid response, and point-of-care (POC) diagnosis. Considering significant advances in SP-based in vitro diagnostics, SP-based immunoassays (SPIAs) would play a critical role in the near future in providing cost-effective mobile healthcare to remote, decentralized, and POC settings. They would be highly beneficial to the developing countries with limited access to basic healthcare services and resources. Various SP-based devices for immunoassays are based on optical detection, electrochemical detection, surface plasmon resonance, lateral flow assays, flow cytometry, and microscopy. Numerous SPIAs have been developed, using innovative diagnostic platforms and technologies, which enable the detection of biomarkers, biomolecules, metabolites, parasites, and pathogens. The coming years will witness an exponential growth in SPIAs and their increased market penetration and outreach. However, several concerns such as private health data and cloud services still need to be tackled.

The immobilization of antibodies on sensing platforms is the first critical step in the development of immunoassays with excellent bioanalytical performance. The ideal immobilization strategy should be leach-proof, rapid, and less labor-intensive to provide high detection sensitivity and specificity without the requirement of high-end skillset and instrumentation. Further, it must be amenable to automation and micro/nanofabrication toward the development high-throughput devices for immunodiagnostics with parallel sensing. Therefore, the surface of most bioanalytical platforms is functionalized to form abundant functional groups for the maximal binding of detection antibodies. Nanoscale characterization techniques provide vital insights into the stability and activity of the immobilized Ab using different immobilization methods. Such nanoscale techniques can be combined with tools from chemistry and molecular biology to improve immobilization strategies. This chapter provides an in-depth overview and insights into various Ab immobilization procedures, surface functionalization chemistries, and surface characterization.

Immunoassays (IAs) play a prominent role in healthcare and industrial settings to provide an early and precise detection of a single or multiple analytes/biomarker in clinical samples. Such useful and vital information enables healthcare and bioanalytical professionals to take appropriate decisions in a timely manner. Under growing concerns for public health and safety, the regulatory has imposed very stringent bioanalytical requirements for IAs. Although industries and in vitro diagnostic (IVD) manufacturers have obeyed most regulatory requirements, the bioanalytical deficiency of several approved IAs has been increasingly noted. Consequently, the bioanalytical method guidelines must be revised toward the development of IAs with minimal bioanalytical shortcomings. The current trend focuses on the development of medical value IAs using the clinical inputs and the end-user evaluation for each development step. This chapter provides an overview of the bioanalytical requirements for IAs, including the critical advances, technical challenges, and trends in this field.

Quartz crystal microbalance (QCM) sensing with high detection sensitivity has been extensively used together with a specific recognition biomolecule for the analysis of disease biomarkers, clinically relevant analytes, pathogens, environmental pollutants, and food contaminants. Many prospective bioanalytical applications at point-of-care settings have been advocated and proven by this sensing approach. This chapter provides an overview of the QCM sensing and its principle of operation together with numerous immunodiagnostic applications. The technical challenges and future prospects in this field are also discussed.

In the Third World, POCT plays an extremely important role in supplying the populations with laboratory medical tests. This chapter describes the preeminent problems within third-world healthcare systems and demonstrates how POCT can also be implemented sensibly within the scope of international development alliances.

This chapter outlines a series of analytical developments trending in POCT systems like miniaturization and parallelization. Focus will also be placed on the importance of smartphones for future applications, primarily in the personalized mHealth sector.

The coming years are anticipated to witness continuous innovation and breakthroughs in immunoassay (IA) formats, bioanalytical platforms, biosensing concepts, detection devices, and complementary technologies. The next-generation IAs, based on highly simplified IA formats, require only minimal process steps and critically reduced sample volumes without any washing steps. They will be fully automated, cost-effective, and equipped with smartphone- or gadget-based mobile healthcare capabilities. Therefore, they can be deployed at centralized, decentralized, remote, and personalized settings with minimal or no requirement for power supply and skilled analysts. The IA trend is strongly inclined toward IA formats to perform multiplex detection of biomarkers for a particular disease and offer clinical diagnosis and monitoring effectively via clinical score. The evolving trend toward smartphone-based IA technologies can extensively extend the outreach of diagnostics by providing personalized mobile healthcare monitoring and management.

The enzyme-linked immunoassay (EIA) has been the most widely used immunodiagnostic format in healthcare, industrial, and bioanalytical settings. The last few decades have witnessed the development of manual and automated EIA formats. Initially, the manual EIA was the predominant format when the field of clinical diagnostics was less advanced. Nevertheless, the enzyme-linked immunosorbent assay, the most used EIA format, is still deployed in resource-limited settings. The trend has now shifted toward automated EIAs, which can be performed in central laboratories using clinical analyzers. Indeed, a wide range of analyzers have become available to suit the customized needs and requirements of various healthcare settings. The recent trend is also strongly inclined toward point-of-care testing using advanced lab-on-a-chip (LOC) formats, rapid EIA strategies, and portable, low-cost readout devices, notably smartphone readers. The advances in complementary technologies, such as mobile healthcare, LOC, multiplex detection, microfabrication techniques, and clinical scoring, are further paving the way to low-cost and analytically superior EIAs

The last two decades have witnessed striking advances in immunoassays (IAs), thanks to innovation and developments in antibody immobilization strategies, rapid diagnostic platforms, novel IA formats, new biosensing concepts, portable detectors, and complementary technologies. These advances have led to the emergence of a wide range of IAs for diversified bioanalytical applications. Most of the initial efforts focused on IAs for central laboratories while the subsequent efforts aimed at decentralized settings. Thereafter, the critical advances in complementary technologies, such as lab-on-a-chip platforms, microfluidics, portable detectors, and novel assay formats, paved the way to several point-of-care IAs. The current trend is strongly inclined toward IA formats for point-of-care testing and personalized mobile healthcare to empower the patients and the general population to monitor and manage their own health. This chapter provides an overview of the critical advances achieved in IAs.

The last two decades have witnessed numerous advances in lab-on-a-chip (LOC) platforms–based immunoassays (IAs). Such developed LOC platforms are diversified and comprise microfluidic chips, paper, lateral flow, cellphone, electrochemistry, and new biosensor concepts. In particular, the development of point-of-care testing devices for developing countries and remote settings has received significant attention. There are novel IA concepts and strategies for prolonged reagent storage and a significant progress in the achievement of next-generation automated, cost-effective, and easy-to-use LOC IAs. Nevertheless, some key technical issues must be addressed for the development of robust and clinically viable LOC-based IAs.

Microelectromechanical systems (MEMS) have come into existence in the 1990s. Microcantilevers are highly simplified MEMS devices that have been used for diversified applications to detect biomolecules, microorganisms, chemicals, and gaseous analytes. For multiplexing, a miniaturized biochip for various analytes has also been demonstrated using an array of microcantilevers. The development of nanocantilevers has further scaled down the technology and considerably improved the detection sensitivity.

Editorial

Human C-reactive protein (CRP) has been reported as an inflammatory biomarker with the highest reference for use in clinical practice. However, the existing analytical techniques are lacking automation and simplicity, as desired for a prospective immunoassay format for point-of-care (PoC) analysis. We have developed an automated magnetic chemiluminescent immunoassay (MCIA) on a mobile analyser for rapid PoC determination of CRP. The MCIA is fully automated after the initial loading of sample and immunoreagents at the inlet ports. The automated protocol involves the transportation of magnetic capture microparticles between adjacent reaction compartments using a set of stationary magnets, a microfluidic polymer disposable and a specific centrifugal protocol. The developed MCIA has a sample-to-answer time of 25 min and hands-on time of approximately 5 min. It detects the entire pathophysiological range of CRP, as desired for clinically-relevant high sensitivity CRP immunoassay format, i.e. 3–81 ng mL−1 in diluted human serum with a limit of detection (LOD) and limit of quantification (LOQ) of 1.5 ng mL−1 and 1.8 ng mL−1, respectively.

This article reveals a rapid sandwich enzyme-linked immunosorbent assay (ELISA) for the highly sensitive detection of human C-reactive protein (CRP) in less than 30 min. It employs a one-step kinetics-based highly simplified and cost-effective sandwich ELISA procedure with minimal process steps. The procedure involves the formation of a sandwich immune complex on capture anti-human CRP antibody-bound Dynabeads in 15 min, followed by two magnet-assisted washings and one enzymatic reaction. The developed sandwich ELISA detects CRP in the dynamic range of 0.3 to 81 ng ml−1 with a limit of detection of 0.4 ng ml−1 and an analytical sensitivity of 0.7 ng ml−1. It detects CRP spiked in diluted human whole blood and serum with high analytical precision, as confirmed by conventional sandwich ELISA. Moreover, the results of the developed ELISA for the determination of CRP in the ethylenediaminetetraacetic acid plasma samples of patients are in good agreement with those obtained by the conventional ELISA. The developed immunoassay has immense potential for the development of rapid and cost-effective in vitro diagnostic kits.

We have developed a highly sensitive immunoassay using graphene nano platelets (GNPs) for the rapid detection of human lipocalin-2 (LCN2) in plasma, serum, and whole blood. It has the dynamic range, linear range, limit of detection, and analytical sensitivity of 0.6 to 5120, 80 to 2560, 0.7, and 1 pg/ml, respectively. It is the most sensitive assay for the detection of LCN2, which has 80-fold higher analytical sensitivity and 3-fold lesser immunoassay duration than the commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kit. The functionalization of microtiter plate (MTP) with GNPs, dispersed in 3-aminopropyltriethoxysilane (APTES), provided the increased surface area that leads to higher immobilization density of capture antibodies. Moreover, the generation of free amino groups on MTP and GNPs by APTES enables the leach-proof covalent crosslinking of anti-human LCN2 capture antibody by its carboxyl groups using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) as the heterobifunctional crosslinker. The anti-LCN2 antibody-bound MTPs were highly stable given that they did not show any significant decrease in their functional activity when stored at 4 °C in 0.1 M phosphate-buffered saline (PBS) for 8 weeks. The developed immunoassay correlated well with the conventional ELISA, thereby demonstrating its high precision and potential utility for highly sensitive analyte detection in industrial and clinical settings.