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Biosensors & Biochips for Sustainable Future

B A S I C L E V E L

Biochips appeared as an innovative microtechnology platform for analysis of bio-molecule in the 1980s.

Biosensors & Biochips: An overview

Biochips appeared as an innovative microtechnology platform for analysis of bio-molecule in the 1980s. A variety of technologies, such as life sciences, information technology, microelectronics and micromechanics, are involved in the underlying technologies. Biochips are considered important potential instruments in modern life science research, medical diagnosis, drug discovery, food safety monitoring and agriculture as high-performance, miniaturized, automated and cost-effective features. Biochips are expected to allow the speed and scope of the analytical process to be dramatically increased and to provide enormous economic value. Further more, in recent years, numerous governments and industrial companies in the world have invested heavily in this sector. Biochip technology is just in its early evolution for now. This is an area which is ever-changing. The future for biochip research and development is bright. There is furious competition in this area.

In biomedical and life science research, the miniaturization of chemical and biomedical laboratory processes on microchips is a rapidly expanding field. Lab-on-chip technologies will bring a lot of benefits over their macro-sized counterparts. In particular, higher surface-to-volume ratios result in reduced chemical requirements, reduced waste, better control, rapid processing and significant ability for parallel processing and process incorporation. Lab-on-chip technologies has the potential to have a significant socio-economic impact. In laboratory medicine, completely integrated microdevices for chemical synthesis and disease diagnosis offer a scientific breakthrough and a paradigm change in chemical processing. The human genome project has made a huge contribution to this technology.

In the field of analytics, biosensors allow major innovations that are both facilitating and facilitated by developments in synthetic biology. The potential of biosensors to identify a wide range of molecules easily and precisely makes them highly important to a variety of industrial, medical, ecological, and science applications. Biosensor design strategies are as numerous as their applications, with major groups of biosensors including nucleic acids, proteins, and transcription factors. Based on the expected use, and the parameters required for optimum performance, each of these types of biosensors has advantages and limitations. Especially, considerations such as ligand specificity, sensitivity, dynamic range, functional range, output mode, activation time, ease of use, and ease of engineering must be considered when choosing the design of the biosensor.

Blueprints for Biosensors: Design & Operation

Biosensors are sensors that transform bio-recognition processes through a physico-chemical transducer into observable signals, with electronic and optical techniques as two main transducers. The creation of biosensors addresses today’s rapidly rising need for clinical diagnostics. A combination of advantages is brought on by the use of biosensors. Biosensors, first, are highly sensitive. This is because biomolecules have a high affinity for their targets, for example, antibodies catch antigens with a dissociation constant at the nanomolar scale, and DNA – DNA interactions are so much stronger than antigen-antibody. Second, biological recognition is typically very selective. The enzyme and substrate are much like a lock and a key, for example. Such high selectivity frequently leads to biosensors that are selective. Third, the production of inexpensive, integrated, and ready-to-use biosensor devices has become relatively easy to develop due to the development of the modern electronic industry. The ability to detect pathogens or perform genetic analysis in hospitals is certainly improved by these biological sensors; more importantly, they are especially useful for small clinics and even point-of-care analysis.

For biosensors with clinical applications, a range of new techniques have been developed. Biosensors are, in general, analytical devices constructed of an element of biological recognition and an optical/electronic transducer. The biological element is responsible for the capture of solution analytes and the transducer transforms the binding event to a measurable signal variation. By the nature of recognition, enzyme-based biosensors, immunological biosensors, and DNA biosensors, could categorize the type of biosensors. In addition, electronic biosensors (electrical or electrochemical), optical biosensors (fluorescent, surface plasmon resonance, or Raman), and piezoelectric biosensors (quartz crystal microbalance) are available depending on the type of transducer.

Electrochemical biosensors

For biological sensing, where electrodes function as either electron donors or electron acceptors, electrochemical techniques are particularly useful. Extensive electrochemical experiments have shown that the Marcus electron-transfer theory also complies with heterogeneous electron transfer between electrodes and surface-confined redox molecules, similar to donor-acceptor pairs in homogeneous solutions. This means that small distance changes in redox surface-confined molecules can cause wide variations in the heterogeneous electron-transfer rates that are supposed to translate into detectable changes of electrochemical signals. Hellinga and co-workers, for example, suggested an electrochemical sensing strategy that exploits protein ligand-mediated hinge-bending motions. A gold electrode was first coated with a self-assembled monolayer (SAM), which gives a versatile platform for site-specific protein immobilization. The maltose-binding protein (MBP) was then bound to the surface of the gold electrode with a particular orientation as the redox reporter group of ruthenium (Ru(II)) is fixed at a certain range above the electrode. As the ligand maltose binds to the active site, the Ru(II) reporter moves away from the electrode by causing a hinge-bending motion of MBP (Figure 1).

Figure 1. Construction and operation of electrochemical biosensor

This maltose-binding induced distance change causes concentration-dependent decreases in electrochemical signals, thus providing a way for maltose to be electronically sensed. The use of this highly generalized sensing approach to detect various analytes with a family of proteins or enzymes undergoing ligand-binding induced conformational changes has also been shown.

Enzyme-based biosensors

The first biosensors ever documented were glucose oxidase (GOD)-based biosensors, established by Clark and Lyons in 1962. Hyperglycemia, a chronically raised concentration of blood glucose, is common for diabetes mellitus. As a result, regular control of their blood glucose concentration is essential for diabetic patients. The benefit of electrochemistry coupled with enzyme catalysis is this biosensor, and its newer versions. An electrode immobilized with GOD was Clark’s biosensor. The oxidized form of GOD interacts with glucose in the presence of glucose and produces gluconic acid and reduced GOD, with two electrons and two protons involved. As dissolved oxygen reacts with reduced GOD, this glucose oxidation also consumes oxygen in the solution, thereby producing hydrogen peroxide and oxidized GOD, and lowering oxygen pressure. As a result, by electrochemically sensing oxygen with a Clark oxygen electrode, the electrode can detect the glucose. This kind of sensor is considered a biosensor of the “first generation.” The Yellow Springs Instrument Company (Ohio, USA) marketed this first-generation biosensor in the 1970s.

The second-generation biosensor substitutes the naturally-existing substrate, oxygen, with small artificial redox molecules that act as redox mediators and exchange electrons between electrodes and enzymes. To improve the sensor efficiency, that is, sensitivity and signal-to-noise ratio, a variety of soluble redox molecules, such as ferrocene, thionine, methylene blue, methyl viologen, were used. These mediators were initially dissolved in a solution. They obtain electrons from the electrodes and then, or vice versa, these electrons are shuttled to the redox center of the enzymes. Immobilized mediators were suggested as a step forward, in order to enhance reagentless biosensors. For example, for hydrogen peroxide, Ruan et al in 1998 documented a reagentlesssolid-state sensor. Gold electrodes were first modified with L-cysteine, and then multilayers of horseradish peroxidase (HRP) were connected by glutaraldehyde to the amine group of cysteine, and thionine was further linked to the enzyme by the same chemical link. As a result, the gold electrode was immobilized by both the enzyme and the mediator, which could detect hydrogen peroxide sensitively in the test solution without further reagent addition. An essential advantage of this configuration of the biosensor is that the mediator is fixed on the electrode surface, thereby preventing the diffusion problem.

An alternative solution that included the use of redox polymers was stated by Heller and co-workers. First, they prepared a doped polymer with Os²⁺complex. This type of polymer functions as a “molecular wire” and exchanges electrons between the enzyme and the electrode. The Os-polymer and glucose oxidase are then co-immobilized on the carbon electrode, which generates a sensitive response to the presence of glucose. They were able to make almost 100 % immobilized enzyme molecules electroactive by using these redox polymers, which contributed to a very high sensitivity method of glucose detection.

The commercialization of the enzyme-based second-generation biosensor was quite successful. In 1987, MediSense was established and the pensized ExactechTM glucose sensors were released. This success has led to a health care revolution for diabetic patients. Instead of traveling to hospitals, they were able to control their concentration of blood glucose at home. The MediSense and later amperometric biosensor systems consist of GOD-coated disposable, screen-printed carbon electrodes and mediators (test strips). The sensor starts to function when a droplet of blood is applied to the test strip and records the amperometric response, which is transformed to a digit displayed on the LCD, indicating glucose concentration.

More recently, by designing a reconstructed GOD enzyme, Xiao et al (2003) published a new glucose biosensor generation. They first prepared apo-GOD free of the cofactor of flavin adenine dinucleotide (FAD), then functionalized and reconstructed a 1.4-nm gold nanoparticle with FAD into the apo-GOD. By using a dithiol monolayer, such a reconstructed enzyme was aligned with gold electrodes (Figure 2). They demonstrated that this artificial enzyme’s electron transfer turnover is as high as 5000 s^-1, approximately 8-fold higher than the normal enzyme (700 s^-1). The gold nanoparticle in this system serves as an electron relay for the electrical wiring of the enzyme’s redox center. In this area, the glucose biosensor established by Xiao et al represents a new path, free from any mediator and highly sensitive. More recently, by using single wall carbon nanotubes (SWNTs) instead of gold nanoparticles, Willner’s group documented a modified version of this sensor and realized a similarly superior efficiency. Although there is still no commercialization of this technology, it is expected that state-of-the-art biosensors will be further improved.

Figure 2 Construction and operation of enzyme – based biosensor

Immunological biosensor

To identify environmental or clinically important targets, immunological biosensors depend on a highly specific immunological system, i.e., antibodies and antigens. In reality, immunological biosensors are a modern variant of the enzyme-linked immunosorbent assay (ELISA), with lower costs, increased speed and convenience of service, and sensitivity that is comparable or even higher.

Among the most common ones are electrochemical immunological biosensors. Two types of immunological biosensors are available. First, the electrode is immobilized with a capture antibody, which catches a particular target antigen. Via a secondary antibody tagged with redox molecules or enzymes, signal transduction is achieved. Second, an electrode antigen is immobilized, which detects specific antibodies.

Ju and co-workers established a carcinoembryonic antigen (CEA) amperometric immunological biosensor. Thionine and HRP-labeled CEA antibodies were co-immobilized on a glassy carbon electrode crosslinked with glutaraldehyde. In the solution, which was coupled to the electrode reaction of thionine, HRP catalytically reduced hydrogen peroxide, leading to a catalyzed signal. The redox center of HRP was partly blocked by catching CEA, leading to attenuation of amperometric signals.

Rusling and colleagues have recently taken advantage of SWNTs to enhance immunological biosensor performance (Figure 3).

Figure 3. Construction and operation of immunological biosensor

By using metal mediated self-assembly, they designed vertically aligned arrays of SWNTs (SWNT forest) on pyrolytic graphite electrodes. Anti-HSA, using EDC/NHS, was then covalently bound to the carboxylated ends of the SWNT forest. The electrode was further incubated with a secondary HRP-labelled anti-HSA antibody after catching the HSA target. The HSA target in the test solution can be identified based on the catalytic signal of HRP for hydrogen peroxide. The detection sensitivity, which was around 1 nM, was dramatically improved by the use of SWNT forests. This was probably due to the improved reactivity of electron transfer of HRP encapsulated in SWNT forests.

One of the most relevant clinical tools has been immunological assays. However, existing assay methods, such as ELISA, require large and costly instruments as well as well-trained specialists. For the development of cheap, miniaturized, and compact devices, electrochemical methods are well adapted. As a result, the development of electrochemical immunological biosensors to meet field and point-of-care analysis is highly desirable. It is important to mention that the use of disposable screen-printed electrodes could be crucial towards this goal, comparable to glucose biosensors. In order to carry out high-throughput (HTS) assays, it is also important to establish antibody microarrays based on electrochemistry.

DNA biosensors

There has been tremendous scientific and technological interest in the identification of DNA hybridization events. The rapidly growing interest in chip-based clinical diagnosis has particularly demonstrated this significance. Therefore , a variety of techniques, including optical, acoustic and electronic approaches, have been developed over the years. In past decades, fluorescent detection has dominated state-of-the-art genosensors among them . Electrochemical methods, however, which have proven effective in simple chemical species, especially metal ions, have attracted increasingly growing interested in biologically related species detection applications.

The benefits of electronic detection include: 1) electrochemical detection is typically inexpensive thus enabling highly sensitive and rapid screening; 2) several electroactive labels, e.g. metallocenes, are stable and environmentally insensitive, unlike fluorophores that often have “photo-bleaching” problems; 3) appropriate molecular design and synthesis that generate a variety of derivatives, each with a specific redox potential, have made it possible to label ‘multi-color’; 4) The rapidly established silicon industry has paved the way for the mass production of integrated circuits, making electronic detection particularly appropriate and compatible with microarray-based technologies; 5) The exponential growth of interfacial science and technology has unraveled mysteries in the precise control of surface properties that are one of the key barricades in bioelectronic applications.

At moderate applied voltages, DNA itself is electrochemically silent, while significant interferences are predicted at high voltages that cause DNA bases to be oxidized/reduced (. Millan was the first to suggest sequence-selective DNA target detections based on electroactive hybridization indicators that provide electronic signals and double and single-stranded DNA discrimination. “Sandwich” type detections were suggested in an effort to reduce the high background derived from the minor binding of hybridization indicators to ssDNA. A DNA strand possessing an electroactive label has been introduced to act as the signaling molecule in addition to an immobilized DNA probe. Similarly, with nanoparticle probes, Park and collegues in 2002 have developed an array-based electrical DNA detection that demonstrates high sensitivity and selectivity. A technology based on the relatively high oxidation activity of guanine and its cooperation by exogenous redox catalysts was developed by Thorp one year later. The discrimination of ds/ss is obtained by the fact that guanine has relatively low electron transfer reactivity in duplexes, due to the steric effect. In the detection of PCR products, this method is highly sensitive, but relatively poor in discriminating hybridization events. Moreover, this method is only possible on ITO surfaces so far, because the high oxidation potential still excludes the use of gold.

Despite progress, the development of an all-in-one (i.e. reagentless) sensor that specifically signals target capture is still highly important (i.e., obviating further treatment with either signal molecules or hybridization indicators). A viable means to this end is provided by DNA or RNA aptamers. Aptamers are well-structured DNA or RNA that have high affinity and selectivity for particular targets as well as natural enzymes, thus showing superior robustness to fragile enzymes. They have been a very promising tool for therapy and diagnosis. Until then, for virtually any given target, the well-developed in vitro selection was able to produce aptamers. In view of these benefits, oligonucleotide aptamers are predicted to be the biosensing components of the next generation. An easy, organized hairpin-like DNA with an electroactive label (electronic DNA hairpin) was used by Fan and collegues as the building block to define hybridization events (Figure 4).

Figure 4. Construction and operation of DNA biosensor

Hairpin-like DNA was an incredibly fascinating aptamer that forms the basis of homogeneous hybridization recognition of fluorescent “molecular beacons”. The DNA sequence has been designed so that in the absence of targets, this “beacon” is in the near state while it will be “turned on” when it reaches its particular gene target. The presence of the design of the stem-loop in the structure offers an on/off switch as well as a stringency to differentiate against single DNA hybridization mismatch. A thiolated terminus gives a sticky end to the gold surface of this electronic DNA hairpin, while a ferrocene tag transduces electronic signals at the other end. The initial hairpin localizes the ferrocene proximal to the electrode surface, thus allowing interfacial electron transfer. After hybridization, the formation of the linear duplex structure disrupts the hairpin and forces apart the ferrocene and the electrode. This signiﬁcant distance change (up to a few nm) effectively blocked the interfacial electron transfer and leads to the diminution of corresponding electrochemical current signals. This strategy offers the opportunity to identify 10 pM DNA targets. More importantly, such a design takes advantage of integrating within a single surface-confined hairpin structure the capturing part (probe sequence) and the signaling part (electroactive species). In contrast to most previously proposed solid-state DNA sensors, this design is therefore effectively reagentless, i.e. no exogenous reagent is required during the recognition process apart from DNA targets. This provides the basis for the development of a portable, continuous DNA analyzer that may be useful for medical and military applications.

A medium for long-range electron transfer (ET) via its base stacking was suggested as the DNA double helix. Although this issue has been discussed for a long time, Barton and colleagues have proven electrochemically that well-oriented gold electrode DNA films enable long-range electron transfer and that such ET is highly sensitive to base stacking pertubations like mismatches. They found that electroactive intercalators like methylene blue (MB) could be effectively reduced by a fully matched DNA duplex-modified electrode. The existence of only a single mismatch, however, converts the wire-like ET medium into an insulator, totally disrupting the ET between the MB and the electrode. Via cyclic voltammetric or coulometric assays, which form the basis of a rapid DNA mutation screening sensor, such a difference can be easily read. Barton and colleagues also demonstrated that electrocatalysis could improve the sensitivity of this strategy. In solution, the addition of ferricyanide constantly pulls electrons from electrochemically reduced MB, amplifying electron flow through the double helix of DNA. This helps in detecting ~108 molecules of DNA with a 30-μm electrode. They have also developed DNA-based sensors to detect DNA-binding proteins in parallel with DNA detection. Some DNA-binding proteins or enzymes are believed to interact with the base pair stacking of DNA, transforming the double helix of DNA from effective ET wires to insulators. They established a sensitive way to electrically assay a variety of DNA-binding proteins based on the comparable sensing strategy. Crucially, these sensors discriminate successfully against proteins that bind to DNA, but they do not disrupt base stacking. This certainly confirms that the signal cut-off on protein binding is due to the alteration of the ET medium relevant to base stacking.

The future of clinical biosensors

Despite the rapid improvement in the development of biosensors, clinical applications of biosensors are still uncommon, with an exception being the glucose monitor. This is in direct contrast to the critical need for point-of-care testing in small clinics. We assume the specifications below are relevant. First, high sensitivity: Improvement of sensitivity is an ever-lasting priority in the development of biosensors. It is clear that the sensitivity criterion ranges from case to case. For example, because glucose levels are high in the blood, one does not need very high sensitivity for glucose detection. This is basically part of the reason why glucose monitors have been successful. However, in many situations, in order to meet the requirements of molecular diagnostics and pathogen detection, it is very important to establish highly sensitive biosensors with optimum single-molecule detection. Second, high selectivity: In the application of biosensors, this may be a significant barricade. Most of the biosensors mentioned in the literature function very well in laboratories, but in real test samples, series problems can be addressed. As a result, in order to prevent non-specific surface adsorption, it is important to establish novel approaches to surface modification. Third, multiplexing is crucial for saving assay time, which is particularly important for laboratory or clinic assays. It is therefore important to establish arrays of high-density electrodes as well as electrochemical instruments that can conduct a large number of assays simultaneously. Forth, in order to increase portability, it is essential to create miniaturized biosensors, thus satisfying the field and point-of-care test requirements. Fifth, it is appropriate to integrate and highly automate an ideal biosensor. A solution to this goal is offered by current lab-on-a-chip technologies (microfluidics). We can expect all these features to be integrated into successful biosensors in the future, and can easily detect minute targets within a short period of time.

Blueprints for Biochips: Design & Operation

The term “biochip” has taken on several meanings. Any device or component introducing biological materials, either extracted from biological species or synthesized in a laboratory on a solid substrate can be considered to be a biochip in the most generic sense. In practical terms, however, both miniaturizations, usually in microarray format, and the possibility of low-cost mass production are often involved in biochips. The electronic nose or artificial nose chip, the electronic tongue, the Polymerase chain reaction chip, the DNA microarray chip (gene chip), the protein chip and the biochemical lab-on-a-chip are some examples which meet these qualifications. In the gene chip and the protein chip, the most dynamic biochip research has been done.

Much attention has been paid, in particular, to biochips integrating conventional biotechnology with semiconductor processing, micro-electro-mechanical systems (MEMS), optoelectronics and digital signal and image acquisition and processing.

Thousands of genes and their derivatives (i.e., RNA and proteins) in a given living organism are generally assumed to act in a complicated and coordinated manner that creates the mystery of life. Traditional methods in molecular biology, however, typically operate on a “one gene in one experiment” basis, which assumes that the throughput is very limited and it is difficult to achieve the “whole picture” of gene function. A new technology, called the DNA microarray, has gained considerable attention among biologists in the last decades. This technology aims to measure the whole genome on a single chip so that, meanwhile, researchers can get a better picture of the interactions between thousands of genes.

A gene or DNA chip corresponds to a two-dimensional array of small reaction cells (100 x 100 μm each) produced using high-speed robotics on a solid substrate. A silicon wafer, a thin sheet of glass, plastic, or a nylon membrane could be the solid substrate. Trillions of polymeric molecules from a particular sequence of single strand DNA fragments are immobilized in each reaction cell (Figure 5).

Figure 5. A schematic illustration of a gene chip

DNA fragments may be either short (about 20 to 25) base sequences (A, T, G, and C) or longer complementary DNA strands (cDNA). In each cell, the unique sequence of bases (e.g. CTATGC…) is preselected or configured depending on the expected use. The probes are also called recognized sequences of single-strand DNA fragments immobilized on the substrate. Double-strand DNA fragments are formed when unknown fragments of single-strand DNA samples, called the target, react (or hybridize) with the probes on the chip, where the target and the probe are complementary according to the base pairing rule (A paired with T, and G paired with C). The target samples are often labeled with tags, such as fluorescents, dyes, or radio-isotope molecules, to facilitate the diagnosis or analysis of the hybridized chip. Each is labeled with its own distinguishable tag when the targets contain more than one type of sample. This type of DNA microarray chip provides a platform where, based on the size of the array, the unknown target or targets can theoretically be defined with very high speed and high throughput by matching the components involved in the research and development of biochip technology with tens of thousands of different types of probes through hybridization in parallel, and the related technical disciplines are indicated in Figure 6. Fundamentally, biochip technology is interdisciplinary; it is important that scientists and engineers from different disciplines cooperate synergistically to push this novel technology from a lab interest to practical devices and systems.

Figure 6. The components and the associated technical disciplines involved in the R&D of biochip technology

DNA microarray

With regard to the property of the arrayed DNA sequence of known identity, there are two variants of the DNA microarray technology:

Type I: probe cDNA (500~5,000 bases long) is immobilized using robot spotting to a solid surface such as glass and exposed either separately or in a mixture to a set of targets. This strategy, “traditionally” referred to as DNA microarray, is largely known to be developed at Stanford University.

Type II: in situ (on-chip) or by conventional synthesis accompanied by on-chip immobilization, an array of oligonucleotide (20~80-mer oligos) or peptide nucleic acid (PNA) probes is synthesized. The array is exposed to hybridized, labeled sample DNA and determines the identity/abundance of complementary sequences. This method, “historically” named DNA chips, was established at Affymetrix INC., which sells its photolithographically manufactured products under the Genechip trademark. Oligonucleotide-based chips are developed by several companies using alternative in-situ synthesis or depositioning technologies.

Depending on the type of molecule immobilized, biochip is made primarily in two formats. CDNA arrays are also referred to as biochips containing PCR products of 200 base pairs to 2KB size immobilized along the length of the molecule by covalently cross linking to the surface of the array. Alternatively, oligonucleotide probes may either be synthesized in situ on the array, or covalent links to the termini may be fixed by pre-synthesized oligos. Genechip engineering includes several different parts, such as manufacturing, sample preparation and hybridization of the target sequence, hybridization results detection, design of the oligonucleotide probe, and hybridization image analysis, and various applications, as seen in Figure 7.

Figure 7. Several important aspects related to Genechip technology

First, according to a particular target, many relevant gene sequences will be selected from the DNA database (single nucleic polymorphism for a specific genes, differential expression for a given group of genes or mutation identification). By determining the sequence and length of each probe and its exact position on the chip, a series of unique oligonucleotide probes will be designed based on the selected sequences. With the spotting method or on-chip synthesis, the synthesis of the DNA microarray can be carried out. The target genes are usually necessary for fluorescence to be amplified and labeled. Selection of appropriate PCR primes, optimization of amplification, and hybridization conditions will be needed in most cases. For the hybridization outcomes on a gene chip, there are several different detection strategies. A traditional approach is a fluorescent detection. For the treatment of such a large amount obtained from a gene chip, data analysis based on the fluorescent images and database configuration is required. The use of PCR products corresponding to the genes as probe molecules is a common platform for preparing microarrays. Biological sources of cDNA libraries offer an effective template for PCR amplification of the probes. This platform is therefore referred to as cDNA Arrays.

The use of oligonucleotide probes instead of PCR products has several benefits. First, they are typical of similar length and can be created in such a way that they have similar properties of hybridization. Second, they may be configured to hybridize against the specific gene region; the PCR product also allows cross-hybridization between homologous genes disturbing members of the same gene family’s gene expression profiles. Furthermore, the need for tedious PCR amplification of the probe molecules is eliminated by oligonucleotide arrays and decreases the risk of error due to clone handling and contamination during transfer.

Microfluidic chip

In recent decades, microfluidic technology has been rapidly developed and provides multitudinous applications in the life sciences. Thanks to the distinct benefits provided by system miniaturization, the microfluidics revolution arose, including high analytical efficiency, increased sensitivity, improved analytical performance, fast multiplexing parallelization, the ability to manage and process reduced reagent volumes and dramatically reduced instrumental footprints.

Microfluidics could simultaneously offer analytical efficiency and high throughput capability as a miniaturization technology, without the lack of accuracy and automation. Microfluidics technology is not only a powerful tool for the fast screening and study of drug development during the drug application process, but also for its miniaturized devices to lower costs and reagent consumption.

Significant progress has been made in developing drug screening components and systems based on microfluidics over the past few years. For drug screening, different types of microfluidic chips are used to improve screening efficiency and decrease costs. Several common types of chip technology are described in the following paragraphs.

Figure 8. Application of microfluidic chip in drug screening.

Droplet microfluidics – In order to perform experiments in continuous or segmented flow, droplet microfluidic technology utilizes liquid droplets compartmentalized by an immiscible fluid as nanoliter to picoliter separate reaction vessels. Such methods show significant advantages, such as reduced sample usage, improved reaction speed and increased efficiency and reproducibility, handling incredibly small volumes with robust composition control.

Based on the sequential process droplet array technique, droplet microfluidic methods may be used for drug combination screening (Figure 8A), to screen various dosing combinations and administration lengths, and to refine the optimum minimally consumed dosing regimen that is important for combined disease situations.

Organ-on-chip – The detailed regulation of microscale structure and flow enables the precise modeling of the microscale structures of organ tissue to be built. Organs-on-chips are biomimetic systems that are micro-engineered and represent essential functional units of living human organs. The functions of multiple organs and tissues, such as the liver, kidneys, lungs and intestines, have been replicated as in vitro models to date. These systems could be used as in vitro models that enable complex biological processes to be simulated and pharmacologically modulated.

The organ chip simulates the human body’s basic processes, by using a number of cells to create a biomimetic chip with similar physiological functions on the chip’s unique structure, which is comparable to the disease’s actual external environment than the typical single-cell culture model.

In order to replicate the complex microarchitecture of cancerous tissue, a microsystem that allows co-culture of breast tumor spheroids with neighboring cells in a compartmentalized 3D microfluidic system has been established to help create the anti-breast cancer drug screening platform (Figure 8B). For the study of cancer cell migration and anti-cancer drug screening, co-culture of multiple tissues with a microfluidic system could be used.

Other microfluidic chips for drug screening – There are several other technologies applied to microfluidic chips for drug screening, in addition to the above-mentioned existing methods, which extend the ideas of researchers. The most significant and integrated aspect of drug development in most pharmaceutical and several biotechnology industries around the world has been an outstanding HTDS system.

With the use of open-access microfluidic tissue array systems and cell microarray chips, various concentrations and combinations of drugs can be screened. Various configurations and arrays of small culture chambers may be generated by the different design of the chip. Concentration gradient generators can provide an effective liquid concentration gradient, and these devices have been implemented and combined with microfluidic technologies by several research groups to optimize HTDS systems. The diffusive microfluidic mixers in Figure 8C could also recognize a fully automatic HTDS.

Cell array platforms are built using polydimethylsiloxane (PDMS) material in certain microfluidic channels for drug screening. The structure of the chips, though, is difficult and has some disadvantages, such as expensive silicon molds and biomolecular absorption. As a natural extracellular matrix (ECM), poly (ethylene glycol) diacrylate (PEGDA) hydrogel has identical mechanical properties and water content. PEGDA microfluidic hydrogels have been commonly used for cell encapsulation and are permeable to compounds such as water, biomolecules and chemicals. In order to research the combinatorial treatment effect of two drugs, microfluidic devices made of these types of materials and combined with 3D brain cell culture technologies were used (Figure 8D).

The allure of the microfluidic chip is that to perform various functions, it can have multiple designed structures and can be combined to extend its application range with different devices and testing equipment. However, considerable design, manufacturing and optimization efforts are needed. Each model has unique features of its own. It is used not only for drug screening, but for drug testing as well.

Protein microarray

In the research of proteins, protein microarrays are useful tools in an unbiased, high-throughput manner, as they allow up to thousands of individually purified proteins to be characterized in parallel. This technology’s adaptability has made it possible to be used in a wide range of applications, including the study of proteome-wide molecular interactions, the analysis of post-translational alterations, the discovery of new drug targets, and the evaluation of pathogen-host interactions. In addition, the technology has already been shown to be effective in profiling the specificity of antibodies, as well as in identifying new biomarkers for autoimmune diseases and cancers in particular.

Proteins are complex biomolecules with a large spectrum of structures and functions, and as such, studying them in a high-throughput manner is a challenge. Three primary types of protein microarrays exist: functional, analytical, and reverse phase. Functional protein microarrays are assembled in a high-through-put manner with proteins purified/synthesized, allowing hundreds and even thousands of different proteins to be examined in parallel with their biochemical properties. In order to detect or measure complex biological samples, analytical protein microarrays use affinity reagents immobilized on the array. Finally, complex biological samples immobilized on the array are used by reverse phase protein microarrays which use affinity reagents for identification.

Functional protein microarrays are more capable of identifying weak interactions, more flexible for low-abundance proteins, and more capable of analyzing crude samples such as serum, compared to other methods, such as mass spectrometry. In terms of differences in proteome coverage, protein lengths, and production pipelines, several different types of functional protein microarrays have been produced to date.

Development of the Functional Protein Microarray – The whole set of proteins that can be expressed by a genome is the proteome. Typically, the creation of a purified proteome microarray requires the assembly of a genome-wide set of open reading frames (ORFs) cloned into an expression vector, encoded protein expression in cells, high-throughput individual protein purification, and protein immobilization on a microarray.

Application of Yeast Proteome Microarrays – For the profiling of proteome-wide molecular interactions, functional protein microarrays, especially purified proteome microarrays, are useful and enable detailed, unbiased screening. Researchers have used functional protein microarrays in fundamental research to study protein-protein interactions, protein-lipid interactions, protein-DNA interactions, protein-cell/lysates, small molecule binding, protein-RNA interactions, and PTMs, such as acetylation, SUMOylation, glycosylation, ubiquitylation, phosphorylation, and methylation (Figure 9A-9G). We summarize representative studies in Table I based on the research applications seen in Figure 9.

Figure 9. Application of Functional Protein Microarray

Applications of biochips

The most emerging applications of biochips are included in Table 1.


Biochip in food	Biochip detecting genetically modified organisms (gmo’s) in food.
Biochip in diagnostics	DNA biochip which revolutionise the way the medical profession performs tests on blood.
Biochip in Tuberculosis epidemic	Biochip technology expected to help combat the new variety of drugresistant strains of the disease.
Biochip in cancer	Biosensor chip technology provides quick and easy access to critical information regarding DNA damage from cancer-producing compounds and aid in early detection of colon cancer.
Biochip in cancer	DNA chips which find genetic differences between people who respond to a drug and those who do not, starting in Phase II, or mid-stage, clinical trials.

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Boon EM, Livingston AL, Chmiel NH, et al. 2003. DNA-mediated charge transport for DNA repair. Proc Natl Acad Sci U S A, 100:12543–7.
Boon EM, Salas JE, Barton JK. 2002. An electrical probe of protein-DNA interactions on DNA-modiﬁed surfaces. Nat Biotechnol, 20:282-6.
Brazill SA, Kim PH, Kuhr WG. 2001. Capillary gel electrophoresis with sinusoidal voltammetric detection: A strategy to allow four-“color” DNA sequencing. Anal Chem, 73:4882–90.
Burgstaller P, Girod A, Blind M. 2002. Aptamers as tools for target prioritization and lead identification. Drug Discov Today, 7:1221–8.
Choi Y, Hyun E, Seo J, et al. 2015. A microengineered pathophysiological model of early-stage breast cancer. Lab Chip, 15: 3350–3357.
Cooper MA, Dultsev FN, Minson T, et al. 2001. Direct and sensitive detection of a human virus by rupture event scanning. Nature Biotechnol, 19:833–7.
Dai Z, Yan F, Yu H, et al. 2004. Novel amperometric immunosensor for rapid separation-free immunoassay of carcinoembryonic antigen. J Immuno Methods, 287:13–20.
Das, H.K. 2005. “Functional Gernomics using Microarrays Technology.” Text book of Biotechnology, pp .1276-1288, Wiley Dreamtech Publisher.
Drummond TG, Hill MG, Barton JK. 2003. Electrochemical DNA sensors. Nat Biotechnol, 21:1192–9.
Fan C, Plaxco KW, Heeger AJ. 2003. Electrochemical interrogation of conformational changes as a reagentless method for the sequence-speciﬁc detection of picomolar DNA. Proc Natl Acad Sci U S A, 100:9134–7.
Fan C, Plaxco KW, Heeger AJ. 2005. Biosensors based on binding-modulated donor-acceptor distances. Trends Biotechnol, 23:186–92.
Fan Y, Nguyen DT, Akay Y, et al. 2016. Engineering a brain cancer chip for high-throughput drug screening. Sci. Rep., 6: 25062.
Fritz J, Cooper EB, Gaudet S, et al. 2002. Electronic detection of DNA by its intrinsic molecular charge. Proc Natl Acad Sci U S A, 99:14142–6.
Gao Z, Binyamin G, Kim H-H, et al. 2002. Electrodeposition of redox polymers and co-electrodeposition of enzymes by coordinative crosslinking. Angew Chem Int Ed, 41:810–13.
Gaylord BS, Heeger AJ, Bazan GC. 2002. DNA detection using watersoluble conjugated polymers and peptide nucleic acid probes. Proc Nat Acad Sci U S A, 99:10954.
Grifﬁths AD, Tawﬁk DS. 2000. Man-made enzymes – from design to in vitro compartmentalisation. Curr Opin Biotech, 11:338–53.
Heeger AJ. 2000. Nobel Lecture: Semiconducting and Metallic polymers: The fourth generation of polymeric materials [online]. URL: http:// wwwnobelse.
Hook F, Ray A, Norden B, et al. 2001. Characterization of PNA and DNA immobilization and subsequent hybridization with DNA using acoustic- shear-wave attenuation measurements. Langmuir, 17:8305–12.
Hsiung LC, Chiang CL, Wang CH, et al. 2011. Dielectrophoresis-based cellular microarray chip for anticancer drug screening in perfusion microenvironments. Lab Chip, 11: 2333–2342.
Li Z, Su W, Zhu Y, et al. 2017. Drug absorption related nephrotoxicity assessment on an intestine-kidney chip. Biomicrofluidics, 11: 034114.
Lin D, Li P, Lin J, et al. 2017. Orthogonal screening of anticancer drugs using an open-access microfluidic tissue array system. Anal. Chem., 89: 11976–11984.
Liu J, Zhang Y, Jiang M, et al. 2017. Electrochemical microfluidic chip based on molecular imprinting technique applied for therapeutic drug monitoring. Biosens. Bioelectron., 91: 714–720.
Moore CD, Ajala O.Z, Zhu H. 2016. Applications in high-content functional protein microarrays. Curr. Opin. Chem. Biol., 30: 21–27.
Palecek E, Jelen F. 2002. Electrochemistry of nucleic acids and development of DNA sensors. Crit Rev Anal Chem, 32:261–70.
Palecek E. 2004. Surface-attached molecular beacons light the way for DNA sequencing. Trends Biotechnol, 22:55–8.
Park SJ, Taton TA and Mirkin CA. 2002. Array-based electrical detection of DNA with nanoparticle probes. Science, 295:1503–6.
Patolsky F, Lichtenstein A, Willner I. 2001. Detection of single-base DNA mutations by enzyme-ampliﬁed electronic transduction. Nature Biotech, 19:253–7.
Patolsky F, Weizmann Y, Wilner I. 2004. Long-range electrical contacting of redox enzymes by SWCNT connectors. Angew Chem Int Ed, 43:2113–17.
S. Mi, Z. Du, Y. Xu, et al. 2016. Microfluidic co-culture system for cancer migratory analysis and anti-metastatic drugs screening. Sci. Rep., 6: 35544.
Schuster GB. 2000. Long-range charge transfer in DNA: transient structural distortions control the distance dependence. Acc Chem Res, 33:253-60. Sullivan CKO. 2002. Aptasensors–the future of biosensing? Anal Bioanal Chem, 372:44–8.
Stone HA, Stroock AD, Ajdari A. 2004. Engineering ﬂows in small devices: microﬂuidics toward a lab-on-a-chip. Annu. Rev. Fluid Mech., 36: 381–411.
Sugiura S, Hattori K, Kanamori T. 2010. Microfluidic serial dilution cell-based assay for analyzing drug dose response over a wide concentration range. Anal. Chem., 82: 8278–8282.
Taton TA, Mirkin CA, Letsinger RL. 2000. Scanometric DNA array detection with nanoparticle probes. Science, 289:1757–60.
Thorp HH. 2003. Reagentless detection of DNA sequences on chemically modiﬁed electrodes. Trends Biotechnol, 21:522–4.
Umek RM, Lin SW, Vielmetter J, et al. 2001. Electronic detection of nucleic acids–A versatile platform for molecular diagnostics. J Mol Diag, 3:74–84.
Van Hove AH, Antonienko E, Burke K, et al. 2015. Temporally tunable, enzymatically responsive delivery of proangiogenic peptides from poly (ethylene glycol) hydrogels. Adv. Healthc. Mater., 4: 2002–2011.
Whitesides GM, Grzybowski B. 2002. Self-assembly at all scales. Science, 295:2418–21.
Willner I. 2002. Biomaterials for sensors, fuel cells, and circuitry. Science, 298:2407.
Wosnick JH, Swager TM. 2000. Molecular photonic and electronic circuitry for ultra-sensitive chemical sensors. Curr Opin Chem Biol, 4:715–20.
Xiao Y, Patolsky F, Katz E, et al. 2003. Plugging into enzymes: nanowiring of redox enzymes by a gold nanoparticle. Science, 299:1877–81.
Xu H, Wu H, Huang F, et al. 2005. Magnetically assisted DNA assays: High selectivity using conjugated polymers for ampliﬁed ﬂuorescent transduction. Nucleic Acids Res, 33:e83.
Yu CJ, Wan YJ, Yowanto H, et al. 2001. Electronic detection of single-base mismatches in DNA with ferrocene-modiﬁed probes. J Am Chem Soc, 123:11155–61.
Yu X, Kim SN, Papadimitrakopoulos F, et al. 2005. Protein immunosen- sor using single-wall carbon nanotube forests with electrochemical detection of enzyme labels. Mol Biosyst, 1:70–8.

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Benson DE, Conrad DW, de Lorimier RM, et al. 2001. Design of bioelectronic interfaces by exploiting hinge-bending motions in proteins. Science, 293:1641–4.
Boon EM, Ceres DM, Drummond TG, et al. 2000. Mutation detection by electrocatalysis at DNA-modified electrodes. Nature Biotech, 18:1096–100.
Boon EM, Livingston AL, Chmiel NH, et al. 2003. DNA-mediated charge transport for DNA repair. Proc Natl Acad Sci U S A, 100:12543–7.
Boon EM, Salas JE, Barton JK. 2002. An electrical probe of protein-DNA interactions on DNA-modiﬁed surfaces. Nat Biotechnol, 20:282-6.
Brazill SA, Kim PH, Kuhr WG. 2001. Capillary gel electrophoresis with sinusoidal voltammetric detection: A strategy to allow four-“color” DNA sequencing. Anal Chem, 73:4882–90.
Burgstaller P, Girod A, Blind M. 2002. Aptamers as tools for target prioritization and lead identification. Drug Discov Today, 7:1221–8.
Choi Y, Hyun E, Seo J, et al. 2015. A microengineered pathophysiological model of early-stage breast cancer. Lab Chip, 15: 3350–3357.
Cooper MA, Dultsev FN, Minson T, et al. 2001. Direct and sensitive detection of a human virus by rupture event scanning. Nature Biotechnol, 19:833–7.
Dai Z, Yan F, Yu H, et al. 2004. Novel amperometric immunosensor for rapid separation-free immunoassay of carcinoembryonic antigen. J Immuno Methods, 287:13–20.
Das, H.K. 2005. “Functional Gernomics using Microarrays Technology.” Text book of Biotechnology, pp .1276-1288, Wiley Dreamtech Publisher.
Drummond TG, Hill MG, Barton JK. 2003. Electrochemical DNA sensors. Nat Biotechnol, 21:1192–9.
Fan C, Plaxco KW, Heeger AJ. 2003. Electrochemical interrogation of conformational changes as a reagentless method for the sequence-speciﬁc detection of picomolar DNA. Proc Natl Acad Sci U S A, 100:9134–7.
Fan C, Plaxco KW, Heeger AJ. 2005. Biosensors based on binding-modulated donor-acceptor distances. Trends Biotechnol, 23:186–92.
Fan Y, Nguyen DT, Akay Y, et al. 2016. Engineering a brain cancer chip for high-throughput drug screening. Sci. Rep., 6: 25062.
Fritz J, Cooper EB, Gaudet S, et al. 2002. Electronic detection of DNA by its intrinsic molecular charge. Proc Natl Acad Sci U S A, 99:14142–6.
Gao Z, Binyamin G, Kim H-H, et al. 2002. Electrodeposition of redox polymers and co-electrodeposition of enzymes by coordinative crosslinking. Angew Chem Int Ed, 41:810–13.
Gaylord BS, Heeger AJ, Bazan GC. 2002. DNA detection using watersoluble conjugated polymers and peptide nucleic acid probes. Proc Nat Acad Sci U S A, 99:10954.
Grifﬁths AD, Tawﬁk DS. 2000. Man-made enzymes – from design to in vitro compartmentalisation. Curr Opin Biotech, 11:338–53.
Heeger AJ. 2000. Nobel Lecture: Semiconducting and Metallic polymers: The fourth generation of polymeric materials [online]. URL: http:// wwwnobelse.
Hook F, Ray A, Norden B, et al. 2001. Characterization of PNA and DNA immobilization and subsequent hybridization with DNA using acoustic- shear-wave attenuation measurements. Langmuir, 17:8305–12.
Hsiung LC, Chiang CL, Wang CH, et al. 2011. Dielectrophoresis-based cellular microarray chip for anticancer drug screening in perfusion microenvironments. Lab Chip, 11: 2333–2342.
Li Z, Su W, Zhu Y, et al. 2017. Drug absorption related nephrotoxicity assessment on an intestine-kidney chip. Biomicrofluidics, 11: 034114.
Lin D, Li P, Lin J, et al. 2017. Orthogonal screening of anticancer drugs using an open-access microfluidic tissue array system. Anal. Chem., 89: 11976–11984.
Liu J, Zhang Y, Jiang M, et al. 2017. Electrochemical microfluidic chip based on molecular imprinting technique applied for therapeutic drug monitoring. Biosens. Bioelectron., 91: 714–720.
Moore CD, Ajala O.Z, Zhu H. 2016. Applications in high-content functional protein microarrays. Curr. Opin. Chem. Biol., 30: 21–27.
Palecek E, Jelen F. 2002. Electrochemistry of nucleic acids and development of DNA sensors. Crit Rev Anal Chem, 32:261–70.
Palecek E. 2004. Surface-attached molecular beacons light the way for DNA sequencing. Trends Biotechnol, 22:55–8.
Park SJ, Taton TA and Mirkin CA. 2002. Array-based electrical detection of DNA with nanoparticle probes. Science, 295:1503–6.
Patolsky F, Lichtenstein A, Willner I. 2001. Detection of single-base DNA mutations by enzyme-ampliﬁed electronic transduction. Nature Biotech, 19:253–7.
Patolsky F, Weizmann Y, Wilner I. 2004. Long-range electrical contacting of redox enzymes by SWCNT connectors. Angew Chem Int Ed, 43:2113–17.
S. Mi, Z. Du, Y. Xu, et al. 2016. Microfluidic co-culture system for cancer migratory analysis and anti-metastatic drugs screening. Sci. Rep., 6: 35544.
Schuster GB. 2000. Long-range charge transfer in DNA: transient structural distortions control the distance dependence. Acc Chem Res, 33:253-60. Sullivan CKO. 2002. Aptasensors–the future of biosensing? Anal Bioanal Chem, 372:44–8.
Stone HA, Stroock AD, Ajdari A. 2004. Engineering ﬂows in small devices: microﬂuidics toward a lab-on-a-chip. Annu. Rev. Fluid Mech., 36: 381–411.
Sugiura S, Hattori K, Kanamori T. 2010. Microfluidic serial dilution cell-based assay for analyzing drug dose response over a wide concentration range. Anal. Chem., 82: 8278–8282.
Taton TA, Mirkin CA, Letsinger RL. 2000. Scanometric DNA array detection with nanoparticle probes. Science, 289:1757–60.
Thorp HH. 2003. Reagentless detection of DNA sequences on chemically modiﬁed electrodes. Trends Biotechnol, 21:522–4.
Umek RM, Lin SW, Vielmetter J, et al. 2001. Electronic detection of nucleic acids–A versatile platform for molecular diagnostics. J Mol Diag, 3:74–84.
Van Hove AH, Antonienko E, Burke K, et al. 2015. Temporally tunable, enzymatically responsive delivery of proangiogenic peptides from poly (ethylene glycol) hydrogels. Adv. Healthc. Mater., 4: 2002–2011.
Whitesides GM, Grzybowski B. 2002. Self-assembly at all scales. Science, 295:2418–21.
Willner I. 2002. Biomaterials for sensors, fuel cells, and circuitry. Science, 298:2407.
Wosnick JH, Swager TM. 2000. Molecular photonic and electronic circuitry for ultra-sensitive chemical sensors. Curr Opin Chem Biol, 4:715–20.
Xiao Y, Patolsky F, Katz E, et al. 2003. Plugging into enzymes: nanowiring of redox enzymes by a gold nanoparticle. Science, 299:1877–81.
Xu H, Wu H, Huang F, et al. 2005. Magnetically assisted DNA assays: High selectivity using conjugated polymers for ampliﬁed ﬂuorescent transduction. Nucleic Acids Res, 33:e83.
Yu CJ, Wan YJ, Yowanto H, et al. 2001. Electronic detection of single-base mismatches in DNA with ferrocene-modiﬁed probes. J Am Chem Soc, 123:11155–61.
Yu X, Kim SN, Papadimitrakopoulos F, et al. 2005. Protein immunosen- sor using single-wall carbon nanotube forests with electrochemical detection of enzyme labels. Mol Biosyst, 1:70–8.