Development and Applications of Portable Biosensors

Bio-recognition Element

Antibody-based sensors, also known as immunosensors, are based on the specificity of an antibody–antigen reaction to produce a change in the transducer signal. Both polyclonal and monoclonal antibodies can be used, and the suitability depends mainly on their specificity. Antibodies can be directly immobilized on the surface of the transducer by covalent bonding through amino, carboxyl, or aldehyde groups. Antibodies can also be attached to the surface of magnetic beads to perform immunomagnetic separation and detection. Antibody-based biosensors with various transducing methods have been reported.^8–11 Enzyme-based biosensors^12–14 are based on using enzymes that are specific to the biomolecules under detection to catalyze the generation of a product that can be quantified by a transducer. Most of the enzymes used in biosensors are oxidases that react with dissolved oxygen to produce hydrogen peroxide. Nucleic acid–based biosensors ¹⁵ integrate a natural and biomimetic form of oligo- and polynucleotides, and rely on the very strong base pair affinity between complementary sections of nucleotide strands for their high sensitivity and selectivity. In recent years, the use of synthetic oligodeoxyribonucleotides (ODNs) is gaining popularity. End labels, such as thiols, disulfides, amines, or biotin, are incorporated to immobilize ODNs ¹⁶ to the surface of a transducer element. Electrochemical DNA biosensors, ¹⁷ in which the base-pair recognition event is converted into a measurable electrical signal, are being used for the diagnosis of genetic diseases, pathogens, biomolecules of clinical interest, and so on. Cell-based biosensors (CBBs) are based on viable whole cells that are integrated onto the biosensor platform and function as the sensing unit to indicate the presence of a particular analyte or a group of analytes. CBBs can use bacteria, yeast, or higher eukaryotic cells, including vertebrate or mammalian cells. ¹⁸ CBBs are designed to record the deviation from normal cellular activities of the “sensing cells” due to their exposure to an analyte. Unlike the nucleic acid– or antibody-based biosensors, CBBs respond to the toxins in a physiologically relevant manner to provide additional information on the mechanism of action and toxicological outcome of exposures.

Transducer Element

Optical biosensors include surface plasma resonance (SPR) biosensors, adsorption and reflection biosensors, luminescence biosensors, fluorescence biosensors, and optical fiber biosensors. SPR and fluorescence biosensors are the most popular optical biosensors. Fluorescence biosensors use fluorescent labels for signal generation, and the intensity of the fluorescent signal is used to determine the amount of the antigen being detected. ¹⁹ SPR is a direct optical-sensing technique that measures the refractive index change due to bio-specific interactions occurring in the vicinity of a thin metal film surface. ²⁰ SPR biosensors for detecting influenza virus ²¹ and plant virus ²² have been reported. Piezoelectric biosensors consist of mass-sensitive piezoelectric crystals with excitation electrodes. The piezoelectric transducers allow a binding event to be converted into a measurable signal, such as resonant frequency changes. ²³ The most popular transducer design is based on the quartz crystal microbalance (QCM), adapted to the liquid medium, which gives a direct response signal to characterize the binding event between a sensitive layer grafted onto the surface transducer and an analyte to be detected. ²³ QCM has been used for the detection of DNA, protein–ligand interactions, virus capsids, bacteria, and mammalian cells. ²⁴ A microcantilever is another type of mass-sensitive biosensor. The principle of this detection is based on the change in the mechanical response of a cantilever due to molecular adsorption on the functionalized cantilever surface. The sensitivity of a microcantilever is high enough to detect a single virus particle. ²⁵ Electrochemical biosensors can be classified into amperometric, potentiometric, and impedance biosensors based on the electrical parameters they measure. Potentiometric biosensors use ion-selective electrodes (ISEs) and ion-sensitive field-effect transistors (ISFETs) to measure the change in electric potential due to the accumulation of ions resulting from an enzyme reaction. ²⁶ Amperometric biosensors measure the change in electrical current (typically, in the nanoampere to microampere range) due to the oxidation/reduction process of an electroactive species. The working electrodes are usually a noble metal or a screen-printed layer covered by a bio-recognition element. ²⁷ The amperometric biosensors are often used for analytes such as glucose, lactate, ²⁸ and sialic acid. ²⁹ In addition, the detection of biological agents such as Bacillus cereus and Mycobacterium smegmatis, ³⁰ the serological diagnosis of Francisella tularensis, ³¹ pesticides, and nerve agents ³² has been reported. Potentiometric biosensors measure the potential difference generated across an ion-selective membrane separating two solutions at virtually zero current flow. A potentiometric sensor array ³³ for investigating the cytotoxicity of hydroquinone to cultured mammalian V79 cells has been reported. Impedance biosensors are based on the combined measurement of the resistive and capacitive properties of the targets in response to a small-amplitude sinusoidal excitation signal.^34–35 Impedance detection involves measuring the change in impedance caused by the binding of analytes to receptors (antibodies, DNA, proteins, etc.) immobilized on the surface of electrodes, ³⁶ change in the conductivity of the medium caused by the growth of bacteria, ³⁷ bacterial cells captured on an electrode surface using dielectrophoresis, ³⁸ and change in the ionic concentration of a medium caused by activity of enzyme used as labels. ³⁹ Calorimetric biosensors are based on measuring the heat of a biochemical reaction at the sensing element. These biosensors consist of temperature sensors with immobilized biomolecules. Once the analyte comes into contact with the biomolecules, the reaction temperature is measured. The total heat produced or absorbed is proportional to the molar enthalpy and the total number of molecules in the reaction. The change in temperature is proportional to the analyte concentration. Calorimetric biosensors have been used for food, cosmetics, pharmaceutical, and other analyses. For example, a microcalorimetric sensor has been used for L-malic acid determination ⁴⁰ in various types of food.

POCT Users and Settings

A detailed description of various POCT users and settings has been published. ⁴³ The simplest POCT with the largest commercial market is self-testing by patients in a home setting for self-assessment and referral. Self-monitoring for blood glucose is one of the oldest applications, with the earliest patents for glucose strips being filed in 1963. ⁴⁵ Some other examples of self-testing include testing for pregnancy and HIV. In some countries such as the United Kingdom, patients are allowed to monitor their own international normalized ratio (INR) to adjust their oral anticoagulation therapy. ⁴⁵ Community POCT usually refers to testing conducted for triage and referral by trained health workers such as pharmacists ⁴⁵ in pharmacies, village workers, and paramedics ⁴³ in a community such as aged care facilities. For example, pharmacies in the United Kingdom and Switzerland have diabetes screening programs that include blood tests and lifestyle advice for an early and cost-effective identification of patients with diabetes and their referral to a general practitioner. Some other examples of testing in the community include testing for malaria, HIV, and dengue. POCT for outpatients in clinics by health care providers (doctors and nurses) is performed for diagnosis and treatment. POCT devices can include a combination of RDTs and handheld instruments. POCT in clinical setting can include testing for HIV, malaria, syphilis, dengue, and Strep A. POCT in peripheral labs is performed by lab technicians for diagnosis, treatment, and monitoring. POCT devices can include not only RDTs but also molecular tests, enzyme-linked immunosorbent assay (ELISA), and microscopy. POCT in peripheral labs can include testing for TB, HIV, malaria, hepatitis B virus, Clostridium difficile, CD4 cell counts, methicillin-resistant Staphylococcus aureus (MRSA), flu, urinary tract infections (UTIs), and viral loads. POCT can also occur in hospital settings ⁴⁶ for diagnosis, treatment, and monitoring. Tests are performed by hospital staff in emergency rooms, operating rooms, and intensive care units to eliminate the need to perform the testing in laboratories. For example, rapid HIV testing can be performed by hospital staff in labor wards ⁴⁷ to reduce mother-to-child transmission.

The cost of POCT devices can make them unaffordable in many countries, and health care providers may receive incentives from private laboratories for ordering tests instead of performing a POCT. The lack of proper guidelines and policies based on strong evidence of the validity and advantages of POCT can also be a barrier to the implementation of POCT. The validation of a POCT device requires strong regulations to ensure that substandard and poor-quality RDTs do not reach the market. A good infrastructure that includes power supply, refrigeration, and waste disposal units at the care centers is necessary for implementation of certain POCTs. A shortage of staff with the required knowledge and training to perform POCT with quality assurance can hinder the implementation of POCT. It may not be possible to claim POCT expenses from insurance providers when these tests are performed in community or home settings. A lack of awareness among health workers and care providers regarding the latest and advanced POCT options available may lead to patients being sent to laboratories for testing. Stigma and a lack of confidentiality associated with tests such as those for HIV may discourage the social acceptance of POCT in community settings.

Antibodies, Antibody Alternatives, and DNA-Based Assays

Among the various biorecognition elements, antibody- and DNA-based approaches have been used widely. Antibody-based detection is one of the main analytical techniques and has proven to be successful in specific and high-affinity detection of biological samples. In recent years, engineered antibody fragments, recombinant antibody fragments (rAbs), single-chain-variable fragments (scFvs), and monovalent antibody fragments (Fabs) have evolved from antibody-based detection and are now emerging as credible alternatives. ⁴⁹ These fragments not only retain the targeting specificity of conventional antibodies but also can be produced more economically. Antibody-based probes can be used for detection of specific proteins, toxins, and also whole cells. For example, antibody-based probes have been used for detection of cholera toxin subunit B (CTB), ⁵⁰ a surrogate biotoxin (ovalbumin) in a raw-milk sample, ⁵¹ and microarray immunoassays for simultaneous detection of proteins and bacteria. ⁵² Antibody-based whole-cell detection in microfluidic systems has been demonstrated in combination with the chemiluminescence technique, ⁵³ the impedance technique, ⁵⁴ and various other techniques. Antibodies are relatively easy to use and are widely available. However, they are costly, have poor chemical and physical stability, require animals for their production, and are limited in options for some analytes. In recent years, alternatives to antibody-based approaches such as enzyme–substrate reactions, ⁵⁵ molecularly imprinted polymers, ⁵⁶ aptamers, ⁵⁷ and antimicrobial peptides ⁵⁸ have been developed.

DNA hybridization assays ⁵⁹ provide more sensitive, specific, and rapid detection of target nucleic acids when compared to conventional antibody-based assays. Many microfluidic DNA-based probes coupled with techniques such as fluorescence resonance energy transfer (FRET), ⁶⁰ conductance impedance, ⁶¹ and surface plasmon resonance imaging (SPRi) ⁶² have been reported. Peptide nucleic acid (PNA) is a DNA mimic and has a peptide backbone instead of a sugar phosphate backbone. PNAs have good chemical and thermal stability, ⁶³ resistance to enzymatic degradation, faster hybridization kinetics, and the ability to hybridize at lower salt concentrations. A PNA beacon ⁶⁴ has been designed for detection of 16S rRNA of Escherichia coli in a droplet-based microfluidic device. PNAs, however, are more expensive than DNA probes.

Amplification

PCR ⁶⁵ is an enzymatic assay that plays a very important role in genetic analysis and biochemical research. PCR enables amplification of a specific DNA fragment from a complex pool of DNA by cycling through three temperature steps and creating several million copies of target DNA within a few hours. Microfluidic devices, when combined with the PCR technique, ⁶⁶ could achieve significant reductions in reaction times. One of the main challenges with PCR in microfluidic systems is prevention of sample evaporation. A design approach ⁶⁷ using nonmiscible mineral oil to cover sample was reported to achieve evaporation loss of less than 5% and simultaneously detect multiple pathogens using an oscillatory-flow multiplex PCR. In recent years, the isothermal nucleic acid amplification ⁶⁸ of DNA and RNA has become popular because it does not require as much energy and thermal momentum for temperature cycles when compared to PCR-based systems. Some of the methods for isothermal amplification include loop-mediated isothermal amplification (LAMP), ⁶⁹ helicase-dependent amplification (HDA), ⁷⁰ nucleic acid sequence–based amplification (NASBA), ⁷¹ recombinase polymerase amplification (RPA), ⁷² and rolling circle amplification (RCA). ⁷²

Sample Preparation

Sample preparation steps are crucial to achieve high sensitivity and specificity in any detection technique. The two important approaches are enrichment of target analyte and/or removal of inhibitors. Sample preparation can be complicated with biological fluids such as saliva, blood, and environmental samples. Some of the popular sample preparation techniques in microfluidic devices include dielectrophoresis (DEP), microparticle- and nanoparticle-based approaches, and filters.

DEP is a term that was first introduced by Pohl ⁷³ to describe the translational motion of particles when subjected to nonuniform electrical fields. The DEP force depends on several parameters, such as the dielectric properties, the shape and size of the particle, the frequency of the applied field, and the electrical properties of the suspension medium. A microfluidic device has been developed for continuous DEP fractionation and purification of sample suspensions of biological cells, ⁷⁴ in which the sorted cells are then delivered to two sample collection ports. A variation of DEP using a combination of both positive and negative DEP has been reported to continuously separate and concentrate bacteria ⁷⁵ from cerebrospinal fluid and blood. Insulator-based DEP (iDEP) in a microchannel containing an array of cylindrical insulating structures, ⁷⁶ in which nonuniform fields are produced by using arrays of insulating structures, has been reported for the concentration and fractionation of a mixture of bacteria and yeast cells. Different electrode structures also have been implemented in a three-dimensional (3D) DEP ⁷⁷ to control the electrical field distribution for continuously sorting and concentrating different types of bacteria.

Micro- and nanobeads have high surface-to-volume ratios with low diffusion times and have been applied to capture and separate target analytes for obtaining higher sensitivity and selectivity in detection. Micro- and nanobeads have been widely used for various applications such as nucleic acid enrichment^78–79 and whole-cell enrichment.^54,80–81 A magnetic nanobead amplification-based QCM immunosensor, ⁸² with magnetic nanobeads coated with anti-H5 antibodies for amplification of the binding reaction between antibody and the H5N1 virus, has been developed. Magnetic micro- and nanobeads provide a means for better particle manipulation when compared to the nonmagnetic micro- and nanobeads. A typical protocol for applying magnetic micro- and nanobeads is based on mixing the functionalized beads with the target sample, ⁸³ followed by application of magnetic field to capture the beads and a wash step to remove any unbound target antigen and analyte. For example, an impedance biosensor system based on a combination of magnetic nanobeads coated with avian influenza virus subtype-specific antibody for capture (separation and concentration) of a target virus, followed by impedance measurement of the nanobeads–virus complexes, has been developed. ⁸⁴ A magnetoresistive immunosensor ⁸⁰ for detection of E. coli, which can detect small variations in the magnetic field caused by the conjugation of magnetic beads to previously immobilized antigens on the surface, has been reported.

Microfluidic filters based on physical filtration provide an alternative to magnetic separation for the rapid separation and enrichment of target samples. A microfluidic filter chip ⁵³ with a stepped microchannel configuration has been reported to capture and enable the formation of a sandwich complex of a monolayer of beads, bacteria, and peroxidase-labeled antibodies inside a microchamber. Polyacrylamide membranes with nanopores ⁸⁵ for simultaneous concentration of viral particles and separation of virus–fluorescent antibody complexes was observed to improve the sensitivity and detection time when compared to the use of an electrophoretic immunoassay alone. Microfabricated pillars that were functionalized with an affinity for bacterial cells inside a PCR chip have been applied to detect E. coli in blood samples.

Capillary Flow Platforms

Capillary flow platforms are commonly known as test strips in which the sample liquids are driven by capillary forces without any applied pressure. The liquid flow is controlled by the wettability and feature size of the microstructured substrate. All of the required chemicals are pre-stored within the test strip. For example, capillary flow platforms such as the LF immunoassay or blood glucose test strips are among the most commercially successful microfluidic POCT devices today. Figure 4 (reproduced from Mark et al. ⁸⁷ ) shows a schematic design of a lateral flow test. Typically, the test results are optical signals and are usually implemented as a color change in the detection area that is visible to the naked eye. Capillary flow platforms are widely implemented and are generally inexpensive. However, capillary flow platforms are difficult to adapt to complicated assays, requiring steps such as mixing, dilutions, washing, and so on. In recent years, numerous efforts have been made to overcome these limitations and enable multiplexing on capillary platforms.

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Figure 4.

Schematic design of a lateral flow test: (A) sample pad (sample inlet and filtering), conjugate pad (reactive agents and detection molecules), incubation and detection zone with test and control lines (analyte detection and functionality test), and final absorbent pad (liquid actuation); (B) start of assay by adding liquid sample; (C) antibodies conjugated to colored nanoparticles bind the antigen; and (D) particles with antigens bind to the test line (positive result), and particles without antigens bind to the control line (proof of validity) Reproduced with permission from Ref. ⁸⁷

Applied Lateral Pressure Platforms

Applied lateral pressure platforms are pressure-driven devices in which external pumps or built-in micropumps are applied to drive fluids through the platform. This platform allows a variety of fluidic operations ⁸⁸ such as mixing, valving, metering, and separation. However, the presence of pumps with fluidic connections to the pump leads to large dead volumes.

Applied Transverse Pressure Platforms

Applied transverse platforms are based on transverse pressure to propel or stop the fluid flow. The microchannels and reservoirs are made of soft elastomers that can be squeezed or pinched off by external pressure exerted by fluid such as compressed air flowing in adjacent channels. Figure 5 shows an example of a transverse pressure-driven flow. The material for these platforms is limited to soft elastomers such as polydimethylsiloxane (PDMS). ⁸⁹ It is possible to design large fluidic networks ⁹⁰ on these platforms, and they are suitable for high-throughput applications.

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Figure 5.

Applied transverse pressure platform. A sequential deflection of polydimethylsiloxane (PDMS) membrane due to pneumatic actuation displaces the liquid underneath to generate flow.

Droplet Microfluidics

Droplet microfluidics, ⁹¹ a subset of microfluidics that involves the generation and manipulation of micrometer-sized emulsion droplets on a microfabricated platform, has started to attract greater attention in recent years. Droplet microfluidics makes the need for conventional micropumps redundant in a microfluidic platform. A controlled and rapid mixing of fluids in the droplet reactors ⁹² results in decreased reaction times when compared to continuous-flow microfluidic devices. Droplet microfluidics can also store all the reagents in droplets and eliminate the need for fluidic coupling to external reagent reservoirs. Any scaling up of droplet microfluidic device does not increase device size or complexity, thereby making these devices very suitable for high-throughput analysis for POCT applications. In digital microfluidic devices, unlike the droplet microfluidic devices, it is possible to address each droplet separately in an array of electrodes without any microchannels, and the droplets can be moved based on the electrowetting-on-dielectric principle, making the device suitable for high-throughput assays. A fully integrated and reconfigurable droplet-based “digital” microfluidic lab-on-a-chip for performing a colorimetric enzymatic glucose assay on human physiological fluids has been developed. ⁹³

Centrifugal Platforms and Lab-on-a-CD

Another approach that can eliminate the need for external tubing and pumping systems is a “lab-on-a-CD,” a technique based on fluid flow driven by centrifugal force achieved on a rotating CD platform. ⁹⁴ Based on a rotating disc, centrifugal pumping can generate a wide range of flow rates ⁹⁴ (from less than 10 nl/s to greater than 100 µl/s) depending on the disc geometry, rotational rate (rotations per minute), and fluid properties. Also, the pumping is not very sensitive to the physicochemical properties such as pH, ionic strength, or chemical composition. Centrifugal pumping of a variety of liquids, including aqueous solutions, solvents, surfactants, and biological fluids (blood, milk, urine, etc.), has been demonstrated. The valving in these systems is achieved either by “capillary” valves in which capillary forces can stop fluids until rotationally induced pressure is sufficient to overcome capillary pressure at the burst frequency or by hydrophobic methods. A wide range of fluidic functions such as valving, decanting, calibration, mixing, metering, sample splitting, and separation can be implemented on a lab-on-a-CD. All of these characteristics of a lab-on-a-CD make it suitable for multiplexing and POCT. A fully integrated lab-on-a-CD that can perform both multiple biochemical analysis and sandwich-type immunoassay simultaneously has been developed ⁹⁵ and is shown in Figure 6 (reproduced with permission from Lee et al. ⁹⁶ ). This platform is suitable for POCT because the required sample volume of blood is small (350 µL vs. 3 mL), takes less time (22 min vs. several days), and does not require specially trained operators or expensive instruments to run the biochemical analysis and immunoassay separately. Researchers at Sandia National Laboratories ⁹⁷ have developed SpinDx, a centrifugal platform for conducting simultaneous multiplexed immunoassays and white blood cell counts from a single finger prick of whole blood with a total sample-to-answer time of less than 15 minutes. A centrifugal microfluidic platform for rapid and accurate determination of the concentration of hemoglobin in human whole blood has been reported. ⁹⁸

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Figure 6.

(A) Photograph of a disc. Detection wells on the clinical chemistry side are preloaded with lyophilized reagents. Other chambers for liquid-type reagent are loaded with food dye solution for demonstration. On the right-hand side, the disc design shows the detailed microfluidic layout. The number indicates the order of the LIFM operation. The top half of the disc for the immunoassay part is rotated for easier demonstration. The blue circles with numbers are (NO)-LIFM. The other half of the disc, for the clinical chemistry analysis part, is shown at the bottom. (B) A photo image of the Samsung Blood Analyzer [25 (W) × 35 (D) × 25 (H) cm] (Samsung, Seoul, South Korea). Reproduced with permission from Ref. ⁹⁶

Acoustically Driven Flows

Surface acoustic waves (SAWs) traveling over the surface of substrate to propel a liquid droplet in the wave propagation direction have been applied to generate, propel, mix, and break up liquid droplets. ⁹⁹ SAW platforms enable enhanced mixing and centrifugation within individual droplets on a microscale. ¹⁰⁰ However, due to the open configuration of SAWs, for application such as PCR that involve a heating step, sample evaporation might be an issue.

Strip-Based/LF POCT

Strip-based/LF tests using capillary forces are the most commercially successful POCT and can be divided into two categories: qualitative tests that can identify the presence or absence of an analyte in the sample, and quantitative tests that provide concentration of the analyte using a readout device. A popular example of a qualitative strip-based/LF test is the pregnancy test. Pregnancy test strips typically are based on an immunoassay in which the presence or absence of a specific protein such as gonadotrophin (hCG) ⁸⁶ is detected based on the interaction of antibodies immobilized on the substrate with antigens in the sample. A test strip for blood glucose level measurements is one of the most popular quantitative LF tests. A drop of blood is placed on a test strip, and capillary action moves the sample to a region of the strip where an enzyme such as glucose oxidase is embedded. The reaction of glucose with glucose oxidase is detected electrochemically in a handheld reader by amperometric detection of the peroxide reaction product, ⁸⁶ and the current is proportional to the blood sugar level in the sample. LF testing has evolved in recent years to include more sensitive bio-recognition elements such as nucleic acid hybridization, ¹⁰¹ resonance-enhanced absorption, ¹⁰² chemiluminescence, ¹⁰³ and silver-enhanced gold nanoparticle labels. ¹⁰⁴ A lot of research is now focused on simplification of LF devices to their highly simplified ingredients: patterned filter paper impregnated with ingredients, the so-called bioactive paper that forms the basis of paper-based analytical devices (µPADs). Recently, paper has been functionalized as a substrate to construct microfluidic devices for use in rapid diagnostics. ¹⁰⁵ A patterning of paper into regions of hydrophilic channels separated by hydrophobic barriers provides four basic capabilities ¹⁰⁵ on a single µPAD: (1) the distribution of sample into spatially segregated regions to enable multiple assays simultaneously; (2) the ability to move the sample based on capillary action without the need for any pumps; (3) the capability to handle small-volume samples such as tears, saliva, urine from neonates, and drops of blood from finger sticks; and (4) easy disposal of hazardous wastes because these devices can be incinerated. The fabrication process is simple, and fabrication material for these devices is usually a cheaper material such as cellulose fiber. Figure 7 (reproduced from Mao et al. ¹⁰⁶ ) shows a µPAD with a multilayer 3D fluidic network formed by stacking layers of patterned paper and double-sided adhesive tape that has been demonstrated for glucose and protein assays.

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Figure 7.

Three-dimensional (3D) paper microfluidic channel network and multiplex detection. Reproduced with permission from Ref. ¹⁰⁶

Handheld and Desktop POCT

Handheld and desktop POCT meets the requirements of multistep tests with automated sample processing, assay steps, and detection. Handheld and desktop POCT platforms use microfluidic technologies ranging from centrifugal fluidics to electrokinetic flows. Typically, desktop platforms use a set of microfluidic cartridges for various types of tests. The popular handheld analyzer i-STAT ¹⁰⁷ (Abbott, Abbott Park, IL), as shown in Figure 8A (reproduced with permission from Chin et al. ¹⁰⁷ ), uses separate fluidic cartridges for blood gases and electrolytes, lactate, coagulation, hematology, and cardiac biomarkers. The operation of i-STAT ¹⁰⁸ begins with introducing sample in the form of whole blood into the cartridge, which is then sealed and inserted into the analyzer. The various processing steps in i-STAT are completely automated to perform amperometric or potentiometric detection on the thin-film biosensor array. Epocal (acquired by Alere in 2010; Alere, Waltham, MA) has developed a portable blood chemistry analyzer, as shown in Figure 8B (reproduced with permission from Chin et al. ¹⁰⁷ ). Fluid manipulation and acutation are achieved using a combination of electroosmotic and pneumatic pumps and capillary flow. Epocal uses self-contained cards called Flexcards with fluidic circuits.

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Figure 8.

(A) The i-STAT (Abbott) handheld analyzer, (B) the Epocal portable blood chemistry analyzer, and (C) the Abaxis Piccolo Xpress (Abaxis, Union City, CA). Reproduced with permission from Ref. ¹⁰⁷

Molecular Diagnostic POCT

There are two major reasons ⁸⁶ for a growing demand for molecular diagnostic POCT. The first reason is the seroconversion delay, which in the case of many infectious diseases has consequences such as permanent damage or death and the possibility that an undiagnosed carrier will transmit the disease to other people. The second reason is the advantage of increased sensitivity that can be achieved by amplification of nucleic acids by the PCR technique. There are numerous molecular diagnostic POCT platforms that are commercially available or under development. For example, Cepheid’s GeneXpert (Cepheid, Sunnyvale, CA) is a real-time PCR-based DNA analysis system capable of DNA extraction, real-time DNA amplification, and detection. ¹⁰⁹ The system is capable of “sample-to-answer” results from an unprocessed sample in approximately an hour. A disposable cartridge contains all the reagents, cell lysis is carried out by ultrasonic energy, and the platform uses an integrated fluorescence detection system. A barcode system is used to store the test results of each test, and the system has a small footprint with low power consumption. Other examples of commercially available molecular diagnostic POCT include Idaho Technology’s FilmArray ¹¹⁰ (BioFire, Salt Lake City, UT) and the Roche cobas Liat (lab-in-a-tube) System (Roche Diagnostics, Basel, Switzerland). ¹¹¹

Infectious Diseases

Infectious diseases are one of the major causes of illnesses and mortality around the world, particularly in developing countries with limited resources. Early detection of pathogens is essential for preventing, treating, and controlling infectious diseases from gaining pandemic proportions. For example, according to the CDC, the 2014 Ebola epidemic is the largest in history, affecting multiple countries in West Africa, and is a serious concern with efforts being made to detect and prevent further spreading of the disease. A complex combination of greater global movement of people and animals, demographic shifts, ecological changes, climate changes, and changes in animal husbandry practices has led to the emergence of an increasing number of new diseases. ¹¹² In developed countries also, there are numerous challenges with regard to pathogen outbreaks, sexually transmitted diseases, and food safety. Traditional methods of pathogen detection such as cell culture and colony-counting methods, PCR, and immunology-based methods such as immunomagnetic separation are time-consuming and laborious. Portable biosensors with the required sensitivity and specificity can overcome these limitations. A rapid detection of an antigen has a significant effect on the success of disease zoning, control, or eradication. Disease surveillance, when combined with good data collection and management, will provide better decision-making tools and early warning systems for disease emergency vigilance. One of the main requirements in developing a biosensor for infectious diseases is a low detection limit without compromising the selectivity. There are many infectious diseases that can spread very rapidly ¹¹³ before any symptoms are observed, and a biosensor with a capability to detect low levels of antigen at the onset of the disease will be a highly invaluable tool. Portable biosensors used for infectious disease also should be inexpensive and robust with high-throughput capabilities.

More than half of the biosensors applied for detection of pathogens are based on electrochemical transducers. ¹¹⁴ This is mainly because electrochemical biosensors have properties such as low cost, high sensitivity, and independence from solution turbidity, and they easily lend themselves to miniaturization, are adaptable to microfabrication, and have low power requirements and relatively simple instrumentation. ¹¹⁵ An amperometric immunosensor ¹¹⁶ for Newcastle disease antibody in poultry serum samples has been developed for concentrations up to 443.24 ng/ml with a detection limit of 11.1 ng/ml. A impedance biosensor for detection of the avian influenza H5N1 virus ¹¹⁷ at a titer higher than 10³ EID₅₀/ml (EID₅₀: 50% egg infective dose) within 2 h has been developed. A portable amperometric biosensor for the rapid evaluation of Salmonella pullorum contamination in chicken with a detection limit of 100 colony-forming units (CFU) per milliliter in culture media and chicken samples within 10 min for each sample has been reported. ¹¹⁸ A novel potentiometric immunosensor ¹¹⁹ for the detection of hepatitis B surface antigen by means of self-assembly to immobilize hepatitis B surface antibody on a platinum disk electrode based on gold nanoparticles has been developed. A dynamic concentration range of 4–800 ng/ml and a 1.3 ng/ml detection limit were observed, and results were observed to be in agreement with the standard ELISA method. The bioelectric recognition assay ¹²⁰ (BERA) is a CBB method that detects the electric response (membrane potential) of cultured cells suspended in a gel matrix to various ligands that bind to the cell and/or affect its physiology. BERA has been applied for detection of hepatitis C virus ¹²¹ in blood samples to rapidly (assay time 3–5 min) and specifically detect at a concentration lower than 100 pg/ml. Impedance spectroscopy is also one of the electrochemical methods that has been applied widely for pathogen detection. Impedance spectroscopy provides the advantage of label-free/reagentless bioaffinity sensing, ¹²² making it suitable for real-time monitoring. A portable impedance biosensor for detection of multiple avian influenza viruses ¹²³ has been reported. It has been proven that impedance spectroscopy is an efficient method when analyzing antigens suspended in clean buffered solutions. ¹²⁴ However, the detection of antigens in a complex medium such as blood remains challenging. It has been observed that the noise signal from nonspecific adsorption is an issue, and many electrode surface modification approaches ¹²⁵ to minimize noise are being explored in this direction. Similarly, conductometric biosensors based on changes in conductance before and after a biorecognition event are also being applied for rapid detection of various foodborne pathogens ¹²⁶ and achieve lower detection limits. A biosensor for detection of bovine viral diarrhea virus ¹²⁷ that is sensitive at an average concentration of 10³ cell culture infective doses (CCID) per milliliter in artificial blood serum samples has been developed. The application of nanomaterials, such as carbon nanotube modified electrodes ¹²⁸ for electrochemical detection, has enhanced the detection capabilities of these devices for various biomolecules. In recent years, screen-printed electrodes ¹²⁹ have garnered a lot of attention in DNA-, immune-, and enzyme-based biosensors. Screen-printed electrodes are a more economically feasible option for mass production of disposable electrodes.

Optical biosensors also have received a lot of attention for detection of pathogens. Numerous optical detection modes such as absorption, reflection, fluorescence, chemiluminescence, and phosphorescence have been applied for biosensors. However, SPR- and fluorescence-based biosensors are more popular for pathogen detection. The detection limit of optical techniques in combination with optical fiber ¹³⁰ is comparable to that of traditional benchtop analyzers. The Sensata Spreeta SPR sensor (Sensata, Attleboro, MA), a commercially available, low-cost SPR sensor, has been demonstrated ¹³¹ for detection of E. coli O157:H7 in beef, apple juice, and ground beef extract with a detection limit of 10² CFU/ml in 30 min with good specificity (it is nonresponsive to E. coli K12 and Shigella). The Spreeta sensor has also been applied for Campylobacter jejuni in poultry meat ¹³² at a low detection limit of 10³ CFU/ml with a low cross-reactivity with Salmonella typhimurium. A SPR-based immunosensor based on synthetic peptides has been developed for detection of hepatitis A virus ¹³³ in human serum with sensitivity comparable to that of ELISA. A SPR biosensor was applied to study the coronavirus ¹³⁴ of severe acute respiratory syndrome (SARS) with the capability to verify that the N-terminal deleted proteinase dimer adopts a state different from that of the full-length proteinase dimer. SPR provides excellent sensitivity, but the required instrumentation is complex and requires a layer of metal to be deposited on the device, thereby raising cost. Also, SPR suffers from strong temperature dependence, which can be a hindrance for portable biosensors in outdoor conditions. Fluorescence-based immunoassays are also of interest for pathogen detection due to their high sensitivity. ¹³⁵ A fluorescence-based multi-analyte immunosensor ¹³⁶ has been developed for simultaneous analysis of multiple samples with various microorganisms and toxins without significant cross-reactivity. A fluorescence immunosensor for detection of dengue virus ¹³⁷ has been reported. One of the drawbacks of the fluorescence technique is that the required reagents are expensive and reaction times are often longer.

Piezoelectric biosensors such as QCMs and SAWs also have been developed for pathogen detection, even though their performance is not as good ¹³⁸ as that of electrochemical and optical biosensors. A QCM biosensor for foot-and-mouth disease virus based on a self-assembled monolayer of alkane thiol ¹³⁹ has been reported. A piezoelectric-based DNA biosensor for the detection of hepatitis B virus ¹⁴⁰ has been developed by immobilizing a nucleic acid probe immobilized onto a gold electrode via a polyethyleneimine–glutaraldehyde cross-linking process. The sensitivity and reliability of piezoelectric biosensors for pathogen detection are comparable ¹⁴¹ to those of the conventional ELISA method. However, the stability of the sensor surface in biological fluids and crystal regeneration remains a challenge.

Chronic Diseases: Monitoring and Diagnostics

Glucose measurement using handheld glucometers is the world leader in commercial volume for POCT. The production volume of glucose test strips for home use has increased tremendously, approaching 10¹⁰/year, with single production lines making devices at a rate of 10⁶/h using printing and laminating technology. ¹⁴² The science and technology of these devices have been extensively described elsewhere. ¹⁴³ Also available are POCT systems for continuous monitoring of glucose, which can replace the invasive, intravenous electrode with the minimally invasive location of a microdialysis catheter in subcutaneous tissue. ¹⁴⁴ Currently available commercial systems include the Guardian RT from Medtronic (Medtronic, Dublin, Ireland), the Abbott Navigator, and the GlucoDay from A. Menarini Diagnostics (Florence, Italy). For diabetes care, subcutaneous devices monitor glucose continuously and wirelessly communicate with an insulin pump in real time, which allows the insulin dose to be adjusted based on preprogrammed patient-specific algorithms.

Blood gas analyzers (BGAs) for pH, pO₂, and pCO₂ are typically based on electrochemical or optical sensors. Some of the commercially available BGAs include the IRMA TruPoint by ITC Medical (Edison, NJ), the Critical Care Xpress series of instruments from Nova Biomedical (Waltham, MA), the cobas b 221 from Roche Diagnostics, and the RAPIDPoint 400/405 from Siemens Medical Solutions (Erlangen, Germany). An optional configuration of a BGA is the inclusion of a CO-oximetry unit. The CO-oximetry unit is a multiwavelength spectrophotometer that measures the typical absorption spectra of the various hemoglobin (Hb) species to distinguish O₂–Hb from other Hb species and to determine O₂–Hb saturation. ¹⁴⁴ Viscoelastic coagulation testing ¹⁴⁵ is the combined analysis of plasma clotting, thrombocyte function, and fibrinolysis. Some of the commercially available instruments include the ROTEM from TEM International (Munich, Germany) and the Sonoclot series from Sienco Inc. (Boulder CO). Optical aggregometry ¹⁴⁶ can be applied to analyze platelet function in terms of in vitro bleeding time. Examples of commercially available analyzers include the PFA-100 System from Siemens Healthcare Diagnostics (Erlangen, Germany) and the VerifyNow System from Accumetrics (San Diego, CA). The significance of plasma lipid levels to assess cardiovascular risks is very well recognized. Some of the small POCT cholesterol systems include Accutrend Plus (Roche Diagnostics), CardioChek PA (PTS Diagnostics, Indianapolis, IN), and MultiCare-in (BSI, Arezzo, Italy). ¹⁴⁷ MultiCare systems are pocket-sized reflectance photometers in which the color intensity from a chromogen reaction is proportional to the concentration of cholesterol or triglycerides in blood. A drop of capillary blood is applied to the test strip to obtain results for cholesterol and triglyceride assays. Desktop POCT cholesterol analyzers, such as the Abaxis Piccolo Xpress (shown in Fig. 8C and reproduced with permission from Chin et al. ¹⁰⁷ ) and the Alere Cholestech LDX, ¹⁴⁸ are also available. The Biosite Triage MeterPlus ¹⁴⁹ (Biosite, San Diego, CA) has immunoassay test panels for cardiac markers. In women, breast cancer is the most commonly diagnosed cancer and is second to lung cancer as a cause of cancer death. A rapid point-of-care breath test for the detection of breath biomarkers of breast cancer has been evaluated. The BreathLink system ¹⁵⁰ (Menssana Research, Newark, NJ) collects, concentrates, and assays breath volatile organic compounds (VOCs) in approximately 6 min. The BreathLink system also has been reported to identify breath biomarkers of active pulmonary TB. ¹⁵¹ An ultrasensitive diagnostic platform called NanoMonitor ¹⁵² (Accu-Chek, Indianapolis, IN) has been developed to enable the rapid label-free analysis of a highly promising class of biomarkers called glycans based on the principle of electrochemical impedance spectroscopy.

Plant Pathogen Detection

Plant pathogen detection is important for detecting bacteria, viruses, and fungi in various settings such as natural landscapes, country borders, greenhouses, and mass production facilities. About 20–30% of the field crops are known to be lost annually due to various diseases. ¹⁵³ It is also important to detect various plant diseases in plantations within forests and other natural environments, and more important for plants that are part of the habitats for wildlife. Among the top 10 bacterial plant pathogens ¹⁵⁴ based on their economic and scientific importance, Pseudomonas syringae and Ralstonia solanacearum are the top two economically important pathogens that cause diseases that infect a range of crops, including potatoes and bananas. A rapid detection of plant pathogens has gained importance due to factors ¹⁵⁵ such as developments in international trade, emerging pathogens with increased resistance to pesticides, increased human mobility, and various new regulations that impose restrictions on the application of toxic chemicals to control the spread of new plant diseases. Early on-site detection of plant pathogens with portable biosensors will enable the design of strategies to control the spread of diseases and will also help the study of disease epidemiology. ¹⁵⁶ The agricultural sector today requires biosensors for detection of multiple potential pathogens or newly emerged pathogens ¹⁵⁷ without necessarily knowing the plant diseases affecting that particular crop group. The development of portable and rapid plant pathogen biosensors would be of value to users from various backgrounds, including individual growers, regulatory agencies, exporters, and researchers. ¹⁵⁸

The traditional plant pathogen detection technique ¹⁵⁶ involves interpreting visual symptoms of disease, which is followed by pathogen diagnosis using microscopy techniques to confirm the data. However, this approach cannot be applied for pathogen diagnosis before the symptoms start appearing. Also, this approach requires analysis from plant pathologists and taxonomists, and is not suitable for on-site diagnosis of plant pathogens. There is a strong demand for developing new techniques for biosensors that would allow rapid and on-site diagnosis of plant pathogens by even unskilled users. The current techniques for plant pathogen detection using portable biosensors can be classified into direct and indirect techniques.

Direct techniques detect the pathogen itself and include immunological techniques using antibody–antibody alternatives and molecular techniques using nucleic acid probes. Among the direct techniques, immunoassays constitute a majority of commercial biosensors for plant disease diagnosis. Molecular techniques mainly include ELISA ¹⁵⁹ and PCR-based methods. Cepheid SmartCycler, ¹⁶⁰ a portable, real-time PCR platform, has been applied for the detection of Phytophthora ramoru, which is known to cause sudden oak death.

Indirect techniques rely on detecting only the effects of the pathogen on the physiological response of the plant. Indirect techniques ¹⁶¹ include spectroscopic/imaging methods and VOC detection methods. Spectroscopic/imaging methods include fluorescence spectroscopy, visible-infrared spectroscopy, and hyperspectral imaging. Fluorescence spectroscopy ¹⁶² relies on the spectral signature to monitor physiological stress levels in plants by analyzing fluorescence at various wavelengths. The spectral signature of a diseased plant ¹⁶³ is influenced by the effect of the disease on plant processes such as chlorophyll degradation, photosynthesis, and so on. Thermal properties of diseased plants change, and this aspect has been applied in infrared thermography ¹⁶⁴ to detect local temperature changes due to plant defense mechanisms in tobacco leaves. X-ray imaging ¹⁶⁵ has been applied for detection of fungal disease in wheat. The presence of pathogens in a plant changes the VOC produced by the plant and can be sensed using electronic nose devices with integrated gas sensors and a pattern identification system. Electronic nose–based biosensors have been applied for diagnosing diseases in wheat ¹⁶⁶ and pear ¹⁶⁷ plants. Indirect techniques rely on comparison between healthy and unhealthy plants, and this frequently requires a recalibration of the biosensor for each plant. Also, indirect methods provide only an indication of whether a disease is present without detecting the disease-causing pathogen.

DNA sequencing is another approach that can be applied for pathogen diagnosis. Genome analysis of plant pathogens ⁶ provides information about the processes and genes involved in the host colonization and pathogenicity, thereby enabling the identification of unknown plant pathogens. One of the challenges for on-site application of sequencing techniques in portable biosensors for plant pathogens is the data analysis. Data analysis ¹⁶⁸ might be difficult to perform in places where internet and communication networks are not available for sending data from a sequencing device to a central database for further analysis. A detailed review of application of DNA sequencing technologies to future portable biosensors for plant pathogen diagnosis has been published. ⁶

Biological Warfare Agents

The detection of toxic compounds is of great significance not only for human health but also for homeland security against bioterrorism. Many biological and chemical agents such as bacteria, viruses, fungi, and toxins are capable of causing serious damage to both animals and humans. It is also possible for the food and water supplies to be contaminated ¹⁶⁹ during terrorist attacks or military actions with BWAs. The biosensor platforms for BWAs, apart from being sensitive and specific, must also be capable of accurately detecting a wide variety of pathogens, including even modified or previously uncharacterized agents, directly from complex sample matrices. ¹⁷⁰ To meet the requirements of continuous monitoring and rapid detection of various BWAs with high sensitivity and specificity, cost-effective and portable biosensors need to be developed. The majority of the biosensors for BWAs have been developed based on electrochemical transducers, ¹⁷¹ but optical transducers ¹⁷² also have gained importance in recent years. The emergence of nanotechnology with new materials such as nanotubes, nanowires, and nanoparticles has opened new avenues for biosensors for pathogen detection. ¹⁷³ Some of the most common BWAs are mycotoxins, exotoxins from E. coli O157:H7, S. aureus, Bacillus anthracis, botulinum, Listeria monocytogenes, Vibrio cholerae, and Yersinia enterocolitica.

Anthrax disease is caused by B. anthracis, and the need for rapid detection methods ¹⁷⁴ has become critical since 2001, when letters containing the spores were mailed in the United States. A label-free detection of B. anthracis based on DNA probe functionalized QCM biosensors ¹⁷⁵ has been reported with a detection limit of 3.5 × 10² CFU/ml of vegetative cells. A portable, label-free optical biosensor ¹⁷⁶ for quantitative detection of a minimum of 34 spores of B. anthracis within 35 min has been developed. A portable biosensor based on oligonucleotide sandwich hybridization ¹⁷⁷ capable of B. anthracis detection within 15 min and no cross-reactivity for 11 tested organisms has been developed. A technology called CANARY ¹⁷⁸ (cellular analysis and notification of antigen risks and yields) has been developed based on genetically engineered B lymphocytes that emit photons within seconds of exposure to pathogens of interest. Within a few seconds, CANARY can rapidly detect as few as 1000 CFU of B. anthracis spores extracted from seeded nasal swabs, ¹⁷⁸ demonstrating the potential to rapidly screen patients for inhalation anthrax exposure.

Botulinum toxin is an acutely lethal toxin produced by the Gram-positive bacterium species Clostridium botulinum, Clostridium barati, and Clostridium butyrium. ¹⁷⁹ The first immunoassay for botulinum toxin ¹⁸⁰ was based on an optical fiber with capture antibodies immobilized on the surface, and it is capable of a detection limit of 30 pM botulinum toxin A within 20 min. A FRET fluorescence-based biosensor platform ¹⁸¹ for detection of botulin toxin light-chain cleavage has been reported with a detection limit of 0.5 nM of toxin light chain for eight samples simultaneously in 2 h. A fluorescence detection portable platform using fluorescent electroluminescent excitation ¹⁸² has been reported for botulinum neurotoxin A detection between 31 and 62 ng/mL after 2 h of incubation. A SPR biosensor ¹⁸³ has been developed for detection of three botulinum neurotoxin serotypes (A, B, and F) with detection limits between 0.5 and 1 ng/mL in less than an hour. CBBs are emerging as an alternative ¹⁸ to ELISA and immuno-PCR methods by eliminating shortcomings such as the inability of ELISA to differentiate between active or inactive toxins or holotoxin and reduced toxin. Human-induced pluripotent stem cells (hiPSCs) have been applied for detecting botulinum neurotoxin and achieving the quantitative determination of different serotypes. ¹⁸⁴ A novel cell-based potency assay ¹⁸⁵ coupled with sandwich ELISA has been reported for botulinum neurotoxin A detection. A comprehensive review of the trend of CBBs in botulinum neurotoxin detection has been published. ¹⁸⁶

S. aureus is a Gram-positive coccal bacterium that can cause diseases such as pneumonia, mastitis, food poisoning, and toxic shock syndrome to humans and animals. A biosensor based on carboxyl-modified CdSe–ZnS quantum dots and oligonucleotide probe complexes ¹⁸⁷ has been developed to detect toxic shock syndrome toxin-1–producing S. aureus with a detection limit of 0.2 µM for the target DNA. A biosensor based on diagnostic magnetic resonance (DMR-3) ¹⁸⁸ in combination with functionalized magnetic nanoparticles has been developed to detect S. aureus in unprocessed biological samples with a detection limit of 10 CFU in less than 30 min. Moreover, the DMR-3-based biosensor has an interface to facilitate system control and data sharing over wireless networks, which in combination with built-in temperature drift compensation makes it suitable for POCT. A piezoelectric cantilever-based biosensor ¹⁸⁹ for sensitive detection of S. aureus enterotoxin B (SEB) has been reported within a concentration range of 2.5–25 fg/mL in apple juice and milk.

Detection of multiple analytes in a single assay is a challenging task. In addition, rapid detection and differentiation of pathogenic versus nonpathogenic species, viable versus nonviable cells, and active versus nonactive toxins are also required. A sensitive detection platform for multiple pathogens, ¹⁹⁰ including Y. pestis, B. anthracis, and SEB, has been developed; it is capable of detecting SEB and inactivated Y. pestis separately at lower concentrations of 5 pg/mL and10⁶ CFU/mL, respectively. A multiple phage-based magnetoelastic ¹⁹¹ biosensor has been developed for simultaneous detection of B. anthracis spores and S. typhimurium with lower detection limits of 5 × 10³ CFU/mL for both. A multiplexed high-density DNA array has been developed with probe-functionalized microspheres ¹⁹² functionalized with 18 different 50-mer oligonucleotide probes and optical encoding indicators. The DNA array included probe sequences designed for B. anthracis, Y. pestis, F. tularensis, Brucella melitensis, C. botulinum, Vaccinia virus, and Bacillus thuringiensis. The DNA array biosensor was demonstrated to achieve a detection limit of 10 fM within 30 min of hybridization for B. anthracis, Y. pestis, Vaccinia virus, and B. thuringiensis. Research in the development of biosensors for BWAs is continually evolving, and future biosensors will be designed to be portable and allow rapid, accurate, and sensitive detection.