Keywords

1 Introduction

The capability to identify diseases and pathogens via detection of associated proteins, nucleic acids, or other biomarkers can provide biomedical researchers and healthcare professionals with highly detailed knowledge of patients’ conditions, disease pathways, or the presence of infection. However, many of the tests commercially available and universally used such as ELISA, PCR and other bioassays have disadvantages of being time consuming and requiring large amounts of sample materials. The results of these problems range from misdiagnosis of disease, inability to timely detect lethal pathogens, and difficulty in understanding the development and progression of the disease. Thus, there is a strong push towards developing improved diagnostic technologies that would allow for rapid, trustworthy, low-cost and multiplexed screening to detect a wide range of biomolecules. To this end, chemical sensors and biosensors have been widely used as attractive alternatives to the bulky, expensive and complex analytical instruments being used in the healthcare sector [1, 2]. A biosensor is a biomolecular electronic device that translates a biological response into an equivalent recordable signal. This device utilizes sensing biomolecules (enzymes, nucleic acid, antibody, virons), support matrix for biomolecule functionalization (planar metal electrodes, carbon electrodes, nanostructured metal electrodes, metal nanoparticles, organic/inorganic nanoparticles) and signal transducers (electrical, optical, mechanical, magnetic) (Fig. 13.1).

Fig. 13.1
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Schematic of biosensor

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In order to overcome the issues associated with current convention diagnostic techniques, a wide range of new biosensors are being developed. Several of these biosensors rely on nanotechnological platforms [3, 4]. The development of analytical tools and processes utilized to fabricate, measure, and image nanoscale objects has paved a path for fabrication of nanosensors which can interact with extremely small number (or an extremely small concentration) of analyte molecules. These advancements are particularly exciting in the field of point-of-care biosensing, where the demands for low concentration detection with high specificity are enormous. Nanoscale biosensors provide researchers with an unparalleled level of sensitivity and detection, often to the single molecule level. In addition, nanostructuring of the sensor surface have enabled significant enhancement in sensor performance surpassing what has been possible with smooth electrode surfaces [5, 6]. These enhancements include increased surface coverage and favorable orientation of biomolecules, and higher electro-catalytic activity at the electrode surface (in case of electrochemical detection systems) [7]. However, after so many advantages proposed by nanobiosensor platforms, biocompatibility and biofouling (specificity/selectivity in complex media) are the two key issues hindering the progress of these sensor for diseases detection in clinical samples. The non-specific accumulation of biological components such as proteins, lipids, and polysaccharides on the sensor surface is referred to as biofouling [8, 9]. Biofouling is disadvantageous to the sensor performance as it impedes the transport of target analyte onto the electrode surface and hinders the reaction between redox moieties and the electrode. This has been a persistent challenge particularly for electrochemical biosensing devices and has motivated the development of several methods to sustain sensor performance in biofouling conditions. To this end, the fouling of sensor can be addresses by controlling the nanostructure geometry which can act as a bio-filter and only allow analyte of interest to pass through it. As nanobiosensor technology becomes more refined and reliable, it is likely that it will eventually make its way from the lab to the clinic, where future lab-on-a-chip devices incorporating arrays of nanobiosensors could be used for rapid screening of a wide variety of analytes at low-cost using small patient samples. The current biosensor market can be broadly divided in two major branches of sensing platforms (1) electrochemical and (2) optical. However, major thrust is shifting towards the use of electrochemical techniques because of their low-cost, portability and multiplexing capabilities. This chapter gives a brief overview of the progress in the field of portable electrochemical biosensing devices, their working principles and further connection of sensors to personalized drug delivery .

2 Electrochemical Methods for Disease Detection

Electrochemical biosensors interface biochemical recognition events (e.g., enzymatic reactions, DNA hybridization, antibody/antigen binding) at molecular level and the corresponding conversion of biochemical event into an electronic signal (Fig. 13.2) in the form of current and voltage [10]. Electrochemical sensors have gained a dominating role in clinical diagnostics owing to their high performance , portability, simplicity, and low-cost [10]. The major benefit of electroanalytical techniques over other detection methods (e.g,. optical, magnetic, and mechanical) is their multiplexing via seamless connectivity with signal processing electronics, without sacrificing the sensor performance [11]. Amperometric, potentiometric and voltammetric techniques are the most widely applied methods in electrochemical analysis and have been utilized for qualitative and quantitative biosensing. Each technique has its own strengths and weaknesses in terms of sensor performance characteristics, however, all of them depend strongly on the electrode, electrolyte, and biomolecule properties. Particularly, the size and morphology of the sensing electrode and the fabrication method used can be very influential on the voltammetric response of the system. The electrolyte containing the redox marker, buffer constituents and supporting salts, has an important effect on the sensor performance. Many redox markers or mediators (MED) have been utilized for signal amplification in case of enzymatic biosensors and hybridization indicators in case of DNA biosensors due to their fast electron transfer properties [10]. The first generation of enzymatic biosensors did not rely on the use of external redox markers and thus suffered problem due to non-specific interfering species (Fig. 13.3a). The added redox markers in the second generation of enzymatic biosensors showed faster electron transfer and allowed low potential device operations (Fig. 13.3b). However, they indeed added extra steps to the sensor preparation process. This issue was addressed in the third generation of enzymatic biosensors where the electrode used for biomolecule immobilization itself shows excellent redox properties and provided a way to achieve direct electron transfer from biomolecule to the electrode (Fig. 13.3c). Most of the current commercial electrochemical biosensor belong to either second or third generation schemes. K4[Fe(CN)6] and Ru(NH3)6 are the most widely used redox markers to study enzyme reactions [12,13,14], while, methylene blue (MB) , has been extensively used as DNA hybridization indicator [15].

Fig. 13.2
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Electrochemical cell-setup and possible signal readouts

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Fig. 13.3
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Various mechanisms of electrochemical enzyme biosensors

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The electrochemical reactions of these redox markers at nanostructured electrodes are also dictated by their reaction rates and transport properties. For example, some redox markers such as MB undergo sluggish reactions with the electrode surface; thereby they can travel further into high aspect ratio structures (such as nano-pores, nano-wells) without losing their activity by fast reactions at the electrode surface [5]. On the other hand, other redox markers for example K4[Fe(CN)6] and Ru(NH3)6 react rapidly with the electrode surface because of their high reaction kinetics and therefore lose their activity on the surface without accessing the entire surface areas in case of nanostructured electrodes [12]. The former examples of redox markers are reaction-limited while the latter are diffusion-limited. Thus, the right selection of the redox markers is important for the fabrication of sensitive electrochemical sensor. Taken together, nanostructured electrochemical sensors in combination with biorecognition elements and accompanying selection of redox markers offer a rich design space for developing tunable sensor platforms for disease detection.

3 Role of Micro/Nano Fabrication in Biosensor Development

Micro/nano fabrication is a useful process for constructing miniaturized physical objects with in micrometer to nanometer range [16, 17]. Microfabricated devices have existed for more than three decades with several applications achieving commercial and scientific success. They are being particularly important in the healthcare industry for downscaling the electrode dimensions in order to fabricate miniaturized electrochemical sensing devices. Besides working towards mobilization of the sensing devices the micro/nano fabrication technologies are giving hope to probe the biological systems on the smallest length scales. The commercial sensor industry is continuously advancing towards microfabrication technologies for developing miniaturized sensing electrodes.

Microfabrication uses a sequence of processes to attain the final device, first of them is thin-film growth/deposition on a substrate which can generally be an order of micrometers in thickness. The second is photolithography, which transfers a pattern (made using AutoCAD) onto the substrate. The next etching step creates miniaturized features by selectively removing materials (either thin films or substrate) in defined patterns. The later steps comprise of binding two or more substrates in order to make the final device structure. These fabricated devices are used for biofunctionalization and lead to the final biosensing devices. The details of the microfabrication technologies can be found in the previously published books [18]. Figure 13.4 shows one of the examples for developing an enzyme based sensor using the microfabrication technology. In this report, the researchers developed H2O2 biosensor consisted of hydrogel (Polyethylene glycol, PEG ) microstructures with entrapped horseradish peroxidase (HRP) immobilized on an array of miniature gold electrodes [19]. The micropatterned Au arrays were prepared by first spin-coating a protective layer of positive photoresist on top of the Au-coated glass slide followed by soft-baking at 115 °C. The electrode pattern previously made in AutoCAD was formed on the slides using a mask aligner. During the UV exposure process the exposed regions of photoresist in the mask gets dissolved in the developer solution and leaves the underneath gold region unprotected which can be removed by wet metal etching process and results in the final array pattern. In this way the photolithography can be used to pattern largely varying geometries and dimensions on a single chip.

Fig. 13.4
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Fabrication of Au patterned electrodes using photolithography and deposition of enzyme hydrogels for biosensing (Figure taken from Matharu et al. Anal Chem 2013, 85, 932–93) [19]

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The microfabrication technology confers various advantages on the biomedical industry as it provides scalability to the devices by reducing the system size for point-of-care use. One such example is microfabricated flow cytometers [20,21,22], which employ microfabrication techniques to make miniature flow chambers. Although these devices cannot compete with the power and versatility of conventional flow cytometers, their portability may be useful for point-of-care hematological tests. Additionally, the close relationship between microfabrication and conventional semiconductor fabrication allows one to integrate electronics or electrical components with microfabricated biochips. The microfabricated biochips also offer detection of various analytes simultaneously and thus are useful for multiplexed detection. The microfabrication and microfluidics are also giving a new direction and level to single-cell analysis which is essential for fatal diseases like cancer [23].

4 Commercially Available Electrochemical Biosensing Devices

4.1 Detection from Invasive Fluids

Electrochemical methods have played a pivotal role in detecting the changes that occur during a biorecognition event (e.g. enzymatic reaction, DNA hybridization) and the merging of microfabrication with electrochemical detection has enabled development of various hand-held biosensor devices operating with small amounts of invasive fluid samples such as blood, serum and urine. Substantial progress in electrochemical sensing has led to the development of some of the very popular commercial hand-held glucose analyzers developed by leading diagnostic companies such as Roche Diagnostics, Inc. and Abbott, Inc (Fig. 13.5). ACCU-CHEK developed by Roche Diagnostics, Inc. relies on few microliters of blood sample that undergoes an enzymatic reaction at the sensor surface. Potential difference is applied to the sensor in a programmed sequence that results in biamperometric current data [24].

Fig. 13.5
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ACCU-CHEK diabetes monitor (Image taken from ACCU-CHEK website)

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Abbott, Inc. has developed the world’s first hand-held device (i-STAT) for point-of-care clinical assay of blood analysis employing several electrochemical-based transduction methods (i.e., potentiometric, amperometric, conductometric) (Fig. 13.6). The i-STAT Portable Clinical Analyser™ is a hand-held silicon-based multiple-analyte sensor array which is used to monitor various blood electrolytes (i.e., sodium, potassium, chloride, calcium, pH), gases (i.e., carbon dioxide, oxygen) and molecules (i.e., urea, glucose, hematocrit) [25]. The individual test for blood gases, electrolytes, creatinine and urea are performed using a miniaturized version of traditional electrode technology contained within a single use test cartridge. To switch from one test group to another, one has to simply change to a different cartridge type. Cardiac troponin I and brain natriuretic peptide tests utilize a sandwich type immunoassay with electrochemical detection. Coagulation tests uses a recombinant human thromboplastin with electrochemical detection of the clotting. Measurement of hematocrit (the ratio of the volume of red blood cells to the total volume of blood) is based on plasma conductivity; the electrical resistance of a whole blood sample is proportional to its hematocrit. Further the hemoglobin concentration is calculated from the hematocrit using a standard equation. Likewise, SenDx Medical Inc. (acquired by Radiometer) has developed a compact and portable potentiometric sensor array for determining various ions in the blood.

Fig. 13.6
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i-STAT handheld analyzer (Image taken from Abbott website)

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Oxford Biosensors have also developed a portable hand-held device (Multisense™) for cholesterol quantification. The biosensor consists of disposable test strips (microelectrodes) and utilizes the electrochemical detection strategy. Chemel (Lund, Sweden) have developed a portable biosensor (SIRE biosensor) that is based on enzymatic/amperometric measuring principles. This technology has been used to measure various sugars and alcohols. Sensor Tech. Ltd. (Cambridge, UK) developed an immunosensor (Universal Transducer System) for in vitro diagnostic and biosensor applications. The Universal Transducer System (UTS™) employs a potentiometric detection platform. The multi-array biosensor was fabricated using screen-printing technology based on glassy carbon powder containing tyrosinase and transduction chemistry [26]. The University of Ulster has developed a biosensor for the determination of flavanols using either plant tissue material (polyphenoloxidases or commercial tyrosinase) [27] immobilized on either a carbon paste electrode or screen-printed electrode with modified polypyrrole.

Various organizations have directed their efforts towards developing DNA biochips. For example, GeneOhm Sciences have developed a DNA chip that employs the electrochemical detection platform. Established in San Diego, California, in 2001, GeneOhm Sciences is a company that focuses on molecular diagnostics for a wide range of diseases. Likewise, Motorola Life Sciences Inc. is another organization that has produced an electrochemical-based DNA chip. A number of small companies appear to be making some progress in the development of various hand-held devices.

4.2 Detection from Non-Invasive Fluids

Most of the commercially available biosensors rely on whole blood, urine or serum samples for disease detection. However, reliability on invasive fluids pose hurdles on real-time monitoring which is essential for finding out the onset of a disease. Continuous analyte monitoring is of particular importance in areas such as regular glucose monitoring for diabetes management, fitness monitoring, drug efficacy and real-time detection of pathogens. The sensors employing invasive fluids have limitations regarding continuous availability of these fluids from the patients. To this end, non-invasive biological fluids such as saliva, tears and sweat can serve as useful substitutes for monitoring various analytes. Wearable sensors employing non-invasive bodily fluids for detection have received enormous attention over the past decade [28, 29] in the field of health and fitness monitoring. These miniaturized sensing devices has shifted the diagnostic industry from hospital-based patient care to home-based personalized monitoring making the field of diagnostics cost-effective and user friendly.

In this context, the research group of Dr. Joseph Wang have taken the electrochemical biosensors to a whole new level by using non-invasive biological fluids for detection of several analytes of clinical interest. Recently, Wang and co-workers reported a wearable non-invasive mouthguard amperometric biosensor for detecting lactate [30] (Fig. 13.7). This was acknowledged as the first example of a wearable electrochemical salivary sensor for continuous metabolite sensing. The sensor was fabricated on polyethylene terephthalate (PET) substrate and subsequently affixed to a mouthguard for continuous saliva lactate detection in undiluted human saliva samples. The group is continuously working on non-invasive monitoring of salivary lactate with integrated electronic backbone and wireless transmitter within the mouthguard.

Fig. 13.7
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Mouth guard sensor and its sensing mechanism (Figure taken from Kim et al. Analyst, 139, 1632–1639) [30]

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In an another report, Parviz’s and co-workers have demonstrated biofunctionalization of a PET-based contact lens with glucose oxidase enzyme (GOx) within a Titania sol–gel matrix for continuous glucose monitoring [31].They further developed an amperometric glucose sensors in the form of contact lens with in-built wireless electronics for continuous data transmission [32, 33]. The authors later modified their wireless sensor to incorporate a dual sensor consisting of activated and deactivated GOx for minimizing interference effect [33].The authors studied the effects of ageing, temperature, and biofouling on the sensor response and demonstrated excellent sensitivity, detection limit, and linear range using a polymer eye model.

NovioSense glucose sensor (NovioSense BV) is another potential commercial product being developed in the Netherlands. It consists of a number of parallel coiled wires forming a micro-electrochemical cell with immobilized GOx. The coiled device can be easily placed within the eyelid for continuous wireless glucose sensing. Efforts have also been made in developing tear-based sensors for detecting lactate as an indicator of hypoxic conditions.

5 Importance of Sensing Devices in the Field of Personalized Drug Delivery

Advances in molecular biology have altered the pace of research on the therapeutic development to increase the efficiency of the treatment by the concept of personalized medicine [34, 35]. While there is an increasing trend for personalized medicine, the role of personalized drug delivery systems (DDSs) have not been explored to its full potential. The development of new pharmaceutics for personalized therapy has been increasing rapidly, yet the optimum outcome for the patient requires testing the medicine via a drug delivery system tailored for each patient. Patient-tailored drug delivery becomes more challenging with an increased number of complexities, however; it may improve the overall efficacy of the treatment. The overarching goal of a drug delivery system is to improve the efficacy by releasing the needed amount of pharmaceuticals at a specific rate, time and location over a long period of time while maintaining the therapeutic dose. This can only be achieved by real-time monitoring of the diseases which will further decide the amount of pharmaceutics for a particular patient. To this end, the integration of the drug delivery systems with biosensing devices can be highly advantageous. The incorporation of sensors to track the delivered drug dose volume and flow rate and report to the user in real-time may offer active control over the delivery profile.

Personalized drug delivery is a complex therapy and requires incorporating interdisciplinary studies together to engineer smart multifunctional materials tailored for a bigger impact in patients’ lives . For example, a conductive implant surface can function as an electrode for electrochemical studies, which can be used as a biosensor and also as drug eluting network to release drugs iontophoretically. Incorporating these modalities together into one individualized DDS can enable patient to get the best accuracy in the treatment since only one device itself can detect what is needed and deliver that amount in a controlled manner with a feedback loop. Or integrating materials with imaging capabilities into the drug delivery system can enable imaging while release is being triggered such as ultrasound-propelled [36], light-gated [37, 38] release from porous networks. Moreover, incorporating pharmacogenetic factors with the engineering aspect of these DDSs can have tremendous advancement in the designs. Patients with cancer, diabetes, autoimmune disorders, cardiovascular diseases, infectious diseases or neurological disorders can get immense benefit from these multifunctional integrated sensor and tailored drug eluting devices with improved target site recognition, enhanced imaging during the delivery of multiple loads of drugs at the desired pace of release. However, all these improvements bring not only scientific but also economic and regulatory challenges together. Efforts have been taken by researchers towards this direction show promise for future devices for personalized healthcare [39,40,41]. Sheybani et al. [42] demonstrated a fully integrated implantable electrolysis-based micropump with incorporated electrochemical impedance dosing sensors. This system provided successful delivery, infusion rate control, and dose sensing in simulated brain tissue.

As the degree of personalization increases, development, production, storage and transportation of the system will change and be reflected in the price. Depending on the choice of DDS, a medical procedure may be required for the placement or additional procedures depending on the depletion rate of the drug. From patient’s perspective, the health and economic outcome will be positive in the long run, however it will create a specific and small market for a certain type of devices, which can be a significant barrier to overcome in the industry. Nevertheless, combining both sensor and drug delivery system in one device can reduce the production costs. However, the interdisciplinary approach in today’s fast-paced technology and growing interest in personalized medicine will pave the way to the development of novel individualized drug delivery systems.

6 Conclusions

The progress made in commercialization of electrochemical biosensors for diabetes, pathogens and several other important biomarkers shows significant promise for future biosensor development specially for early and sensitive detection of life threating diseases such as cancer and HIV. Although, the current available sensors mostly utilize invasive body fluids for disease detection, the on-going research by several pioneer groups are showing potential for designing biosensors detecting target molecules in non-invasive biological fluids such as sweat and tears. The next steps in this field would be fabrication of robust sensing platforms that can show detection in smallest possible quantities of the disease related target molecules in biological samples. This can only be achieved by sensor chips on which isolation, amplification and detection of the target can be performed simultaneously. These tools further integrated with the personalized drug delivery systems can be a boon for healthcare industry.