Application of Nanobiosensors in Healthcare

The biological and medical fields have seen great advances in biomolecules. This review is meant to provide an overview of the various types of biosensors and biochips that have been developed for biological and medical applications, along with significant advances over the last several years in these technologies. It also attempts to describe various classification schemes that can be used for categorizing the different biosensors and provide relevant examples of these classification schemes from recent literature.

1. Introduction

The emergence of nanotechnology is opening new horizons for the development of nanosensors and nanoprobes with submicron-sized dimensions that are suitable for intracellular measurements. A biosensor is defined as a ‘‘device that uses specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues, organelles or whole cells to detect chemical compounds usually by electrical, thermal or optical signals’’1. In recent years, many types of biosensors have been developed and used in a wide array of biomedical and other settings. Although it is impossible to survey this entire fast-moving field, this issue presents articles about some of the many types of biosensors and biosensor-based applications to give the reader a sense of the enormous importance and potential for these devices. One of the earliest references to the concept of a biosensor is from Dr. Leland C Clark who created many of the early biosensors in the early 1960’s2 using an ‘‘enzyme electrode’’ for measuring glucose concentration with the enzyme Glucose Oxidase (GOD). The success of single analyte sensors was followed by development of integrated multi-analyte sensors capable of more comprehensive analyses, such as a single instrument for glucose, lactate, and potassium detection. Technical developments in manufacturing enabled the development of miniaturized integrated biosensors for determination of glucose, lactate, and urea in micro samples of undiluted whole blood or plasma. Miniaturization also allowed additional analytical tools to be added to the biosensor, such as chromatography or capillary electrophoresis. The newest generation of biosensors includes miniaturized multi-analyte immunosensor devices with high-throughput capabilities and more than 1000 individually addressable electrodes per square centimeter. These instruments can detect analytes present in the attomole range3. Modern fabrication techniques such as ink-jet printing, photolithography,

 Biosensing principle

Fig.1 Biosensing principle

microcontact printing, and self-assembly continue to contribute to more advanced biosensors, and the next type of devices to emerge may include miniature biosensors with high-density ligands, selfcontained lab-on-a-chip capabilities, and nanoscale biosensors. Biosensor that includes transducers based on integrated circuit microchips are often referred to as biochips. (Fig.1) illustrates the conceptual principle of biosensing process. Biosensors and biochips can be classified either by their bioreceptor or their transducer type ( Fig. 2).

Classification Schemes of Biosensors/Biochip

Fig.2 Classification Schemes of Biosensors/Biochip

2. Nanobiosensors

Nanomaterials are exquisitely sensitive chemical and biological sensors. Nanosensors with immobilized bioreceptor probes that are selective for target analyte molecules are called nanobiosensors. They can be integrated into other technologies such as lab-on-a-chip to facilitate molecular diagnostics. Their applications include detection of microorganisms in various samples, monitoring of metabolites in body fluids and detection of tissue pathology such as cancer. Their portability makes them ideal for pathogenesis of cancer (POC) applications but they can be used in the laboratory setting as well.

2.1. Nanowire biosensors

Since their surface properties are easily modified, nanowires can be decorated with virtually any potential chemical or biological molecular recognition unit, making the wires themselves analyte independent. The nanomaterials transduce the chemical binding event on their surface into a change in conductance of the nanowire in an extremely sensitive, real time and quantitative fashion. Boron-doped silicon nanowires (SiNWs) have been used to create highly sensitive, real-time electrically based sensors for biological and chemical species4. Biotin-modified SiNWs were used to detect streptavidin down to at least a picomolar concentration range. The small size and capability of these semiconductor nanowires for sensitive, label-free, real-time detection of a wide range of chemical and biological species could be exploited in array-based screening and in vivo diagnostics.

2.2 Ion Channel Switch biosensor technologies

The Ion Channel Switch (ICS), a novel biosensor technology of Ambri Ltd (Chatswood, Australia), is based upon a synthetic self-assembling membrane, which acts like a biological switch that detecting the signaling the presence of specific molecules by triggering an electrical current5. This is the basis of the company’s SensiDx System, a nanobiosensor device that has been designed for POC testing in critical care environments in hospitals. By delivering precise and quantitative test results in an immediate timeframe, the SensiDx System reduces the time of emergency diagnoses from hours down to minutes.

2.3. Electronic nanobiosensors

The Biodetect system of Integrated Nanotechnologies ( Henrietta , NY ) works by electronically detecting the binding of a target DNA molecule to sensors on a microchip. The target molecules form a bridge between two electrically separated wires. In order to create a strong clear signal, the bound target molecules are chemically developed to form conductive DNA wires, which are metalized and can be seen by electron microscopy. The bridges, which can be observed by fluorescent imaging techniques, are readily detected by measuring the resistance or other electrical properties of the sensor. These DNA wires ‘turn on’ a sensor much like an on/off switch12. Each chip contains multiple sensors, which can be independently addressed with capture probes for different target DNA molecules from the same or different organisms. A proprietary DNA Lithography process is used to attach capture probes to each of the sensors on the chip. These chips now have billions of capture probes per sensor, which greatly improves sensitivity.

2.4 Viral nanosensor

Virus particles are essentially biological nanoparticles. Herpes simplex virus (HSV) and adenovirus have been used to trigger the assembly of magnetic nanobeads as a nanosensor for clinically relevant viruses6 .The nanobeads have a supramagnetic iron oxide core coated with dextran. Protein G is attached as a binding partner for antivirus antibodies. Anti-HSV antibodies are conjugated directly to nanobeads using a bifunctional linker to avoid nonspecific interactions between medium components and protein G. By using a magnetic field, as few as five viral particles can be detected in a 10 µl serum sample. This system is more sensitive than ELISA-based methods and is an improvement over PCR-based detection because it is cheaper, faster and has fewer artifacts.

2.5 PEBBLE nanosensors

Probes Encapsulated by Biologically Localized Embedding (PEBBLE) nanosensors consist of sensor molecules entrapped in a chemically inert matrix by a microemulsion polymerization process that produces spherical sensors in the size range of 20 to 200 nm7. These sensors are capable of real-time inter- and intra-cellular imaging of ions and molecules and are insensitive to interference from proteins. PEBBLE can also be used for early detection of cancer. PEBBLE nanosensors also show very good reversibility and stability to leaching and photobleaching, as well as very short response times and no perturbation by proteins. In human plasma they demonstrate a robust oxygen sensing capability, little affected by light scattering and autofluorescence 8.

2.6 Optical biosensors

Many biosensors that are currently marketed rely on the optical properties of lasers to monitor and quantify interactions of biomolecules that occur on specially derived surfaces or biochips. Surface plasmon resonance technology is the best-known example of this technology.

2.6.1 Surface plasmon resonance technology

Surface plasmon resonance (SPR) is an optical–electrical phenomenon involving the interaction of light with the electrons of a metal. The optical–electronic basis of SPR is the transfer of the energy carried by photons of light to a group of electrons (a plasmon) at the surface of a metal. The next generation microarray-based SPR systems are designed to help researchers profile and characterize biomolecular interactions in a parallel format. Miniature optical sensors that specifically identify low concentrations of environmental and biological substances are in high demand. Currently, there is no optical sensor that provides identification of the aforementioned species without amplification techniques at naturally occurring concentrations. Triangular silver nanoparticles have remarkable optical properties and their enhanced sensitivity to their nano environment has been used to develop a new class of optical sensors using localized SPR spectroscopy9.

2.6.2 Laser nanosensors

In a laser nanosensor, laser light is launched into the fiber, and the resulting evanescent field at the tip of the fiber is used to excite target molecules bound to the antibody molecules. A photometric detection system is used to detect the optical signal (e.g., fluorescence) originating from the analyte molecules or from the analyte-bioreceptor reaction10. Laser nanosensors can be used for invivo analysis of proteins and biomarkers in individual living cells.

2.7 Nanoshell biosensors

Gold nanoshells have been used in a rapid immunoassay capable of detecting analyte within complex biological media without any sample preparation11. Aggregation of antibody/nanoshell conjugates with extinction spectra in the near infrared is monitored spectroscopically in the presence of analyte. Successful detection was achieved in this system constitutes a simple immunoassay capable of detecting sub-nanogram-per-milliliter quantities of immunoglobulins in saline, serum, and whole blood. Nanoshells are already being developed for applications including cancer diagnosis, cancer therapy, and testing for proteins associated with Alzheimer’s disease. Nanoshells can enhance chemical sensing by as much as 10 billion times. That makes them about 10,000 times more effective at Raman scattering than traditional methods. When molecules and materials scatter light, a small fraction of the light interacts in such a way that it allows scientists to determine their detailed chemical makeup. This property, known as Raman scattering, is used by medical researchers, drug designers, chemists and other scientists to determine the nature of various materials. An enormous limitation in the use of Raman scattering has been its extremely weak sensitivity. Nanoshells can provide large, clean, reproducible enhancements of this effect, opening the door for new, and all-optical sensing applications. Scientists at the Laboratory of Nanophotonics of Rice University (

Houston, TX ) have found that nanoshells are extremely effective at magnifying the Raman signature of molecules, each individual nanoshell acting as an independent Raman enhancer. That creates an opportunity to design alloptical nanoscale sensors–essentially new molecular level diagnostic instruments–that could detect as little as a few molecules of a target substance, which could be anything from a drug molecule or a key disease protein to a deadly chemical agent. The metal cover of the nanoshell captures passing light and focuses it, a property that directly leads to the enormous Raman enhancements observed. Furthermore, nanoshells can be tuned to interact with specific wavelengths of light by varying the thickness of their shells. This tunability allows for the Raman enhancements to be optimized for specific wavelengths of light. The finding that individual nanoshells can vastly enhance the Raman effect opens the door for biosensor designs that use a single nanoshell, something that could prove useful for engineers who are trying to probe the chemical processes within small structures such as individual cells, or for the detection of very small amounts of a material, like a few molecules of a deadly biological or chemical agent12.

3. Clinical applications of biosensors

There has been a great demand for rapid and reliable methods which can be used in biochemical laboratories for determination of substances in biological fluids such as blood, serum and urine, etc. There is also a demand to move clinical analysis from centralized laboratories to a doctor’s clinic and patients self-testing at home. Most of the methods available in the market for rapid detection are based on enzyme electrodes. They provide for a negligible enzyme consumption of <1 mg per sample. The Glucometer GKM 01was the first commercial enzyme electrode based glucose analyzer developed in Europe . It was introduced in 1980 at the Centre of Scientific Equipment of the Academy of Sciences of the GDR. The glucometer is being adapted to the quantification of uric acid, lactate and the activity of acetylcholine esterase. The lipid analyzer ICA-LG 400 from the Japanese company Toyo Jozo is capable of measuring whole group of analytes, namely cholesterol, triglycerides and phospholipids by using enzyme electrodes13. Although biosensors have found immense applications in various fields, their use in health care monitoring is of utmost importance. Recently, measurement of metabolites in media other than blood has a great demand. Such types of measurements are care monitoring is of utmost importance. Recently, measurement of metabolites in media other than blood is important. Where there is a need of continuous monitoring of analytes, such as glucose, urea, etc. therefore invasive biosensing sometimes proves to be very painful for the patients undergoing self-testing. Therefore, the concept of non-invasive testing in sweat, saliva or skin has become popular. Guilbault et al.14 have discussed the work carried out at labs in New Orleans and Rome for development of non-invasive sensors. Recently work on near infra red (NIR) method which is a reagent less system and non-invasive has started gaining interest.

3.1. Ex-vivo monitoring

A few instruments, which are of great help in the treatment of continuous monitoring of diabetes and other metabolites, such as lactate, pyruvate, glucose, etc. have been used for ex vivo monitoring. An artificial pancreas ‘‘betalike’’ (EsaOte Biomedica, Geneoa , Italy ) has been known for continuous measurement of glucose. It takes the blood from a patient vein, dilutes it, dialyze it and re-infuses blood cells into the blood stream and analysis of glucose concentration. Similarly this ex vivo monitoring is used in sports medicine for measurement of lactate concentration. Another glucose sensor (Unitech, Uln) has been introduced as a commercial portable sensor for continuous glucose monitoring. This instrument is helpful for obtaining long-term glucograms. The instrument utilizes an enzyme electrode connected to a wick implanting to equilibrate with subcutaneous fluid. Lipid analyzer (ICA-LG 400) utilizes the enzyme electrode and is useful for determination of triglycerides, cholesterol and phospholipids. An amperometric urea sensor based on pH dependence of the anodic oxidation of hydrazine has been utilized in the glucometer GKM 02. The first enzyme electrode based lactate analyzer was developed in 1976 by LaRosch ( Switzerland) which used Cyt b2 on platinum electrode.

3.2. In vivo monitoring

Several approaches are known for in vivo measurements. One of the approaches consists of an assembly of needles comprising a glucose electrode15 for subcutaneous use and the other approach is microdialysis. Needle sensors have been implanted subcutaneously for several days and results are tele-transmitted to the receiver. The transmitter converts current signal generated by the glucose needle biosensor to a very high frequency audio signal and the receiver demodulates back to a voltage. Microdialysis is a more recent approach to an implantable biosensor and works on the principle of mimicking the function of a blood vessel by implanting a microdialysis probe into the tissue. The probe essentially consists of a thin dialysis tube perfused with a sample (blood). The substances, which are in higher concentration in the extra cellular fluid outside the probe, defuse in as soon as the substances are carried out of the body by the perfusion liquid, the concentration can be determined by coupling it with a biosensor. Other approaches, which have been proposed for in vivo monitoring include enzyme based electrochemical, enzyme based field effect transistor (ENFET), enzyme based thermoelectric, electrochemical and optical approach.

4. Biosensors for health care

4.1. Glucose biosensor

Detection of glucose has been the most studied analyte in diabetic patients. The level of the glucose can be monitored either in vivo or in vitro. The first approach for in vitro study was pioneered by Shichiri et al.15Mascini et al.16 reported an ‘‘artificial pancreas’’ for continuous measurement of glucose. A number of glucose biosensors have been reported which are based on conducting polymers 17, 18, 19 . Ramanathan et al.19 covalently attached glucose oxidase on poly (o-amino benzoic acid) and fabricated the screen printed electrodes made of this material. These electrodes have been shown to be useful for glucose estimation from 1 to 40 mM and stability of about 6 days. The i-STAT portable clinical analyser which is a significant commercially available biosensor, can measure a range of parameters: sodium, chloride, potassium, glucose, blood urea nitrogen (BUN) and haematocrit. The NPL Glucosense developed at the National Physical Laboratory, India is based on the screen printed graphite electrode having a mediator incorporated in the working electrode. The product is available with the Indian markets for the consumers. The sensors are fabricated using thin film microfabrication technology on a disposable cartridge20. Recently, Singhal et al.17 reported that poly (3-dodecylthiophene) /stearic acid /glucose oxidase (P3DT/SA/GOX) Langmuir–Blodgett films based glucose biosensor can be used for at least 35 measurements and was found to be stable upto 40 days.

4.2 Lactate biosensor

Lactate measurement is helpful in respiratory insufficiencies, shocks, heart failure, metabolic disorder and monitoring the physical condition of athletes. Many biosensors have been reported to date21, 22. Two different technologies have been approached for the development of miniaturized systems. Thin film electrodes have been developed, which can be used as either implantable catheter type devices or for in vivo monitoring in combination with microdialysis system23, 24. Secondly, disposable type sensors were developed for the purpose of on-line analysis25, 26. Group at the National Physical Laboratory, India has recently developed a screen printed electrode based lactate biosensor. Li and coworkers28have recently reported the sol–gel encapsulation of lactate dehydrogenase for optical sensing of L-lactate. Such a disposable lactate sensor has a linear dynamic range from 0.2 to 1 mM of lactate and stability of about 3weeks. The sensor was found to have a diminished enzyme activity (about 10%) and leaching of the enzyme from the matrix.

4.3. Urea and creatinine biosensors

Urea estimation is of utmost importance in monitoring kidney functions and disorders associated with it. Most of the urea biosensors available in literature are based on detection of NH4+ or HCO3¯sensitive electrodes28, 29, 30. Osaka et al.31 constructed a highly sensitive and rapidflow injection system for urea analysis with a composite film of electropolymerized inactive polypyrrole and a poly ion complex. Gambhir et al.32 have recently co-immobilized urease and glutamate dehydrogenase on electrochemically prepared polypyrrole/polyvinyl sulphonate for the fabrication of urea biosensor. Singhal et al.28 have recently immobilized urease on poly (N-vinyl carbazole/stearic acid).

4.4. Cholesterol biosensor

Determination of cholesterol is clinically very important because abnormal concentrations of cholesterol are related with hypertension , hyperthyroidism, anemia and coronary artery diseases.

Determination based on the inherent specificity of an enzymatic reaction provides the most accurate means for obtaining true blood cholesterol concentration. Reports on the development of cholesterol biosensors are available33-39. Recently, Vengatajalabathy and Mizutani40 demonstrated an amperometric biosensor for cholesterol determination by a layer-by-layer self-assembly using ChOx and poly (styrenesulfonate) on a monolayer of microperoxidase covalently immobilised on Au-alkanethiolate electrodes. The sensor was found to be responsive even in the presence of potential electrical interferents, L-ascorbic acid, pyruvic acid and uric acid. Kumar et al.33 presented a cholesterol biosensor by co immobilization of cholesterol oxidase and peroxidase on sol–gel films and utilized these films for estimations of cholesterol.

4.5. Uric acid biosensor

Uric acid is one of the major products of purine breakdown in humans and therefore its determination serves as a market for the detection of range of diosorders associated with altered purine metabolism, notably gout, hyperuricaemia and Lesch–Nyhan Syndrome. Elevated levels of uric acid are observed in a wide range of conditions such as leukaemia, pneumonia, kidney injury, hypertension, ischemia, etc. Additionally, as a reducing agent uric acid scavenges free oxygen radicals, preventing their destructive action towards tissue and cells. Various attempts have been made to develop a biosensor for the estimation of uric acid41-47.

4.6. DNA biosensor

DNA biosensors have an enormous application in clinical diagnostics for inherited diseases,

Rapid detection of pathogenic infections and screening of cDNA colonies are required in molecular biology. Conventional methods for the analysis of specific gene sequences are based on either direct sequencing or DNA hybridization48. Because of its simplicity, most of the traditional techniques in molecular biology are based on hybridization. Several immobilization techniques such as adsorption49, covalent attachment50, or immobilization involving avidin–biotin complexation51 were adopted for a DNA probe to the surface of an electrochemical transducer. The transducer was made from carbon52; gold53-55; or conducting polymer56, 57. In the case of a common sandwich assay the signal generating species is an enzyme, such as horseradish peroxidase58. Lund et al.59 linked the tagged DNA to the surface of the microsphere using a suitable reagent. Another effort is the use of microfabrication system and micro mechanical technology to the preparation of DNA samples and their analysis (e.g. DNA chip). Gambhir et al.57 have recently attempted to immobilize DNA on conducting polypyrrole/ polyvinyl sulphonate films and demonstrated the adsorption characteristics. They believed that anion doped polypyrrole undergoes ion exchange with PO4¯ of DNA to facilitating the adsorption. Presently, DNA probes and biosensors have widely attracted attention for diagnosis of various disorders60-63.

4.7. Immuno-sensors

Immuno-sensors are small, portable instruments for analysis of complex fluids and are designed for the ease of use by un-trained personnel, rapid assay and sensitivity comparable to that of ELISA. During the past decade, a number of methods for immunoassay by specific interactions between antibodies and antigens to analyze microorganisms, viruses, pesticides and industrial pollutants have been developed64-67. Immuno-sensors are the analytical systems based on immuno-chemical principles that can automatically carry out estimation of desired parameter. Barnett et al. have detected thaumatin using antibody containing polypyrrole electrodes67. In the recent past, immunoassays have relied on complex indirect enzyme methods in which the resultant product of the enzyme immuno reaction can be measured. Recently, antibodies have been raised against the conducting polymer, carbazole as a hapten, which may react to modulate the polymer electrochemistry.

It has been observed by cyclic voltammetry that the reaction of the antiserum influences the polymer matrix electrochemistry by an amperometric response.

5. Biosensor Transducers

The transducer converts the biochemical interactions into a measurable electronic signal. Electrochemical, electrooptical, acoustical, and mechanical transducers are among the many types

found in biosensors. The transducer works either directly or indirectly.

5.1. Direct detection biosensors

Direct detection sensors, in which the biological interaction is directly measured in real time, typically use non-catalytic ligands such as cell receptors or antibodies. The most common direct detection biosensor systems employ evanescent wave, or surface plasmon resonance (SPR), technology which measures resonant oscillation of electrons on the surface of a metal.

5.2 Indirect detection biosensors

The second class of transducers, indirect detection sensors, relies on secondary elements that are often catalytic elements such as enzymes. Some examples of secondary elements are the enzyme alkaline phosphatase and fluorescently tagged antibodies that enhance detection of a sandwich complex. Dr. Vincent Gau’s and Dr. Omowunmi Sadik’s groups describe the use of electrochemical transducers to measure the oxidation or reduction of an electroactive compound on the secondary ligand in one common type of indirect detection sensor. Other common indirect detection biosensors employ optical fluorescence, measuring fluorescence of the secondary ligand. Dr. Frances Ligler’s group has used optical fluorescence to develop a multi-analyte indirect detection biosensor. Fluorescence resonance energy transfer (FRET), a process where energy from an excited fluorophore is transferred to a neighboring acceptor molecule, is also used for indirect detection2. Finally, light-addressable potentiometric sensors (LAPS) combine electrooptics and electrochemistry for indirect detection, as discussed by Dr. Tatsuo Yoshinobu’s group.

5.3. Integrated biosensors and lab-on-a-chip

Along with the development of better ligands and alternative transducer technologies, there is significant research on manufacturing to produce advanced integrated devices. ‘‘Lab-on-a-chip’’ biosensors contain integrated microfabricated fluidics systems and are designed to perform multi-step high-resolution biological or chemical assays. These devices can contain many channels, allowing for massively parallel biochemical processes and multi-analyte detection3. Many of these devices are fabricated using molding or photolithographic processes developed in the microelectronics industry to create circuits of chambers and channels using composite materials, quartz, silica, or glass chip. As discussed in the article by Dr. Steven Soper’s and Fred Battrell’s groups, microfluidic circuits can be designed to perform many biochemical processes including immunological assays, DNA amplification, manipulation, and analysis, flow cytometry and complex biochemical reactions. Their small dimensions and large relative surface area within the fluidic system reduce reaction times, the amount of reagents, labor, and costs.

Table 1:Various biosensor transducers, principles and applications86

Transducer system

Principle

Applications

Enzyme electrode

Amperometric

Enzyme substrate and immunological system

Conductometer

Conductance

Enzyme substrate

Piezoelectric crystal

Mass change

Volatile gases and vapors

Thermistor

Calorimetric

Enzyme, organelle, whole cell or tissue sensors for substrates,

Products, gases, pollutants, antibiotics, vitamins, etc.

Optoelectronic/wave guide and fiber optic device

Optical pH,

enzyme substrates and immunological systems

Ion sensitive electrode (ISE)

Potentiometric

Ions in biological media, enzyme electrodes, enzyme immunosensors

Field effect transistor (FET)

Potentiometric

Ions, gases, enzyme substrates and immunological analytes

6. Biochip

Biochips are the basis for miniaturized biochemical assays, and offer many advantages over the conventional analytical methods, the most significant of which are: i) a variety of analytes can be investigated simultaneously in the same sample, ii) the required sample quantities are minimal, iii) low consumption of scarce reagents, iv) high miniaturization and v) high sample throughput. These advantages become very evident if we consider the workflow during a typical drug screening process. At the beginning there is the need for a selection of few eligible compounds out of a large variety of molecules for a given purpose. Since the most limiting factor is the ratio between the number of data points per day and the cost per data point, this first mass-selection is best done at a molecular level where DNA-chips and protein microarrays find their most common application. After that the investigations will move to the whole cells, where the Channelomics can deliver very clear informations about the effect of drugs on the physiology of the cells68.

6.1 DNA Biochip

A DNA-chip consists in most cases in a glass or quartz slide acting as a carrier, which is functionalized with an array of probes (features). A single probe contains identical molecules, for this reason it is also called feature. The basic principle of operation of a DNA-chip consists of three main steps: 1) Functionalization, i.e. the immobilization of different DNA sequences onto different positions (probes), 2) Hybridization, bring the analytes in contact with the probes, by flooding the chip with the sample solution and let the hybridization take place, 3) Readout, after washing the chip to remove all non-bound molecules, the array is scanned to detect on which probes a hybridization took place. Depending on the different fields of application, e.g. gene expression for drugs screening, or medical diagnosis of cancer or genetic diseases, the requirements for the DNA-chip may include high sensitivity, wide dynamic range and high specificity. The first two parameters reflect the ability to deliver a correct response at different intensities of hybridization, while the latter shows the efficiency in rejecting analytes with any minimal mismatch68.

6.2 Integrated DNA biochip

The development of a truly integrated biochip having a phototransistor integrated circuit (IC) microchip has been reported by Vo-Dinh and coworkers69, 70. This work involves the integration of a 4 X 4 and 10 X 10 optical biosensor array onto an integrated circuit (Fig. 3). Most optical biochip technologies are very large when the excitation source and detector are considered, making them impractical for anything but laboratory usage. In this biochip the sensors, amplifiers, discriminators and logic circuitry are all built onto the chip. In one biochip system, each of the sensing elements is composed of 220 individual phototransistor cells connected in parallel to improve the sensitivity of the instrument. The ability to integrate light emitting diodes (LEDs) as the excitation sources into the system is also discussed. An important element in the development of the multifunctional biochip (MFB) involves the design and development of an IC electro-optic system for the microchip detection elements using the complementary metal oxide silicon (CMOS) technology. With this technology,

 Schematic diagram of an integrated DNA biochip system

Fig.3 Schematic diagram of an integrated DNA biochip system69

highly integrated biochips are made possible partly through the capability of fabricating multiple optical sensing elements and microelectronics on a single system. Applications of the biochip are illustrated by measurements of the HIV1 sequence-specific probes using the DNA biochip device for the detection of a gene segment of the AIDS virus70. Recently, a MFB which allows simultaneous detection of several disease end-points using different bioreceptors, such as DNA, antibodies, enzymes, and cellular probes, on a single biochip system was developed71. The MFB device was a self-contained system based on an integrated circuit including photodiode sensor arrays, electronics, amplifiers, discriminators and logic circuitry. The multi-functional capability of the MFB biochip device is illustrated by measurements of different types of bioreceptors using DNA probes specific to gene fragments of the Mycobacterium Tuberculosis (TB) system, and antibody probes targeted to the cancer related tumor suppressor gene p53.

6.3 Micro fluidics-based biochip system

The analysis of complex liquid samples for specific components necessitates some means of selective detection. In some cases, discrimination is afforded through spectral characteristics of the components in a complex sample, particularly with infrared, Raman and mass spectroscopes. Otherwise, hyphenated techniques which couple spectroscopic techniques with separation systems are routinely used for the analysis of complex samples (e.g. gas chromatography-mass spectrometry (GC-MS) 72-74, liquid chromatography-mass spectrometry (LC-MS) 75, mass Spectrometry – mass

 Schematic diagram of micro fluidics based biochip system

Fig. 4 Schematic diagram of micro fluidics based biochip system71

spectrometry (MS- MS)76, liquid Chromatography – infrared absorption spectrometry (LC-IR)77,78, ion trap MS79.80, gas chromatography-infrared absorption spectrometry (GC-IR)73,74,77, capillary electrophoresis (CE), etc.81-83.However, with the exception of some innovative MS device-based systems e.g. ion traps79, 80, such instruments tend to be bulky, expensive, labor-intensive, and require skilled operators. Furthermore, individual chromatographic-based separation systems generally have limited applicability in terms of ranges of molecular size and /or functionality. Even with hyphenated techniques, analysis of a complex sample may require multiple pretreatment steps as well as multiple chromatographic systems. Schematic diagram of micro fluidics based biochip system71 (Fig. 4)

7. Future issues in the development of Nanobiosensor and Biochip

New biosensors and biosensor arrays are being developed using new materials, nanomaterials and microfabricated materials including new methods of patterning. Biosensor components will use nanofabrication technologies. Use of nanotubes, Buckminster fullerenes (buckyballs), silica and its derivatives can produce nanosized devices. Some of the challenges will be: Development of real-time non-invasive technologies that can be applied to detection and quantitation of biological fluids without the need for multiple calibrations using clinical samples. Development of biosensors utilizing new technologies that offers improved sensitivity for detection with high specificity at the molecular level. Development of biosensor arrays that can successfully detect, quantify and quickly identify individual components of mixed gases and liquid in an industrial environment. It would be desirable to develop multiple integrated biosensor systems that utilize doped oxides, polymers, enzymes or other components to give the system the required specificity. A system with all the sensor components, software, plumbing, reagents and sample processing are an example of an integrated sensor. There is also a need for reliable fluid handling systems for ‘dirty’ fluids and for relatively small quantities of fluids (nanoliter to attoliter quantities). These should be low cost, disposable, reliable and easy to use as part of an integrated sensor system. Sensing in picoliter to attoliter volumes might create new problems in development of micro reactors for sensing and novel phenomenon in very small channels12.

The size of device to move (levitate) tiny fluid droplet has been reduced by using micron scale diamagnets to create a magnetic micromanipulation chip, which operates with femtodroplets levitated in air84. The droplets used are 1 billion times smaller in volume than has been demonstrated by conventional methods. The levitated particles can be manipulated and positioned with accuracy within a range up to 300 nm. Use of this technology on a lab-on-a-chip would refine the examination of fluid droplets containing trace chemicals and viruses. Even though micro array/biochip methods employing the detection of specific biomolecular interactions are now an indispensable tool for molecular diagnostics, there are some limitations. DNA micro arrays and enzyme-linked immunosorbent assay (ELISA) rely on the labeling of samples with a fluorescent tag—a procedure that is time consuming and expensive. Nanotechnologies can provide label-free detection and are being applied to overcome some of the limitations of biochip technology.

8. Summary

As biosensor technology advances, the range of applications broadens. Biosensors are now being developed for detection of microbial pathogens and their toxins (see articles by Drs. Ligler, Gau, and Sadik and their colleagues), monitoring of glucose and other metabolites, blood analysis (see articles by Drs. Battrell and Gau and colleagues), and other physiological monitoring (see articles by Drs. Yingfu Li and Homola and colleagues), cancer detection (see articles by Drs. Gau, Thundat, Soper, and Battrell and their colleagues), and monitoring. In addition, biosensor technology is being applied to allergen detection, food and biomaterial quality testing, and basic research on molecular interactions. Biosensors offer several advantages over other analytical methods including rapid and even real-time measurements, high sensitivity, selectivity, and specificity even when a complex or turbid sample matrix is used. As the technology advances, producing lab-on-a-chip devices (as described by Drs. Soper and Battrell and their colleagues), these self-contained portable instruments will allow measurements outside the laboratory, in the field or at the bedside. Biochip technologies could offer a unique combination of performance capabilities and analytical features of merit not available in any other bioanalytical system currently available. With its multichannel capability, biochip technology allows simultaneous detection of multiple biotargets. Biochip systems have great promise to offer several advantages in size, performance, fabrication, analysis and production cost due to their integrated optical sensing microchip. The small sizes of the probes (micro liter to nanoliter) minimize sample requirement and reduce reagent and waste requirement. Highly integrated systems lead to a reduction in noise and an increase in signal due to the improved efficiency of sample collection and the reduction of interfaces. The capability of large-scale production using low-cost integrated circuit (IC) technology is an important advantage. The assembly process of various components is made simple by integration of several elements on a single chip. For medical applications, this cost advantage will allow the development of extremely low cost, disposable biochips that can be used for in-home medical diagnostics of diseases without the need of sending samples to a laboratory for analysis.

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Authored by by Murugan Veerapandian

 

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