Biosensors in The Small Scal Methods and Thecnology Trends PDF

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Published in IET Nanobiotechnology Received on 23rd March 2012 Revised on 24th August 2012 Accepted on 4th September 2012 doi: 10.1049/iet-nbt.2012.0005
ISSN 1751-8741
Biosensors in the small scale: methods and technology trends

Sukru U. Senveli1, Onur Tigli1,2
1 2
Department of Electrical and Computer Engineering, University of Miami, Coral Gables, FL 33146, USA Department of Pathology, University of Miami, Miami, FL 33136, USA E-mail: tigli@miami.edu
Abstract: This study presents a review on biosensors with an emphasis on recent developments in the eld. A brief history accompanied by a detailed description of the biosensor concepts is followed by rising trends observed in contemporary micro- and nanoscale biosensors. Performance metrics to quantify and compare different detection mechanisms are presented. A comprehensive analysis on various types and subtypes of biosensors are given. The elds of interest within the scope of this review are label-free electrical, mechanical and optical biosensors as well as other emerging and popular technologies. Especially, the latter half of the last decade is reviewed for the types, methods and results of the most prominently researched detection mechanisms. Tables are provided for comparison of various competing technologies in the literature. The conclusion part summarises the noteworthy advantages and disadvantages of all biosensors reviewed in this study. Furthermore, future directions that the micro- and nanoscale biosensing technologies are expected to take are provided along with the immediate outlook.
Introduction
The remarkable progress of microand nanoelectro-mechanical systems (MEMS and NEMS) in the last two decades followed and coincided by the recent developments in nanotechnology have forced engineers and scientists from many disciplines to collaborate on a diversity of interdisciplinary projects. One of the most promising elds that emerged is biosensors employing the micro- and nanoscale advancements. Especially in the past decade, major breakthroughs have been made in the eld. The associated devices range from nanowire eld effect transistors (FETs) [1, 2] to microcantilevers [35] and whispering gallery mode (WGM) [1, 6] optical sensors, and from optical microring resonators (OMRRs) [7] to surface plasmon resonance (SPR) [6, 8, 9] type detectors and others. Although, the use of nanotechnology and micro- and nanoscale fabrication is relatively new and still ourishing, the history of the eld of biosensors, in a general sense, spans the last half century. Specically, the amperometric enzyme electrode developed by Clark and Lyons in 1962 marks the initiation of the eld. It operated as a glucose sensor and can be regarded the rst ancestor of the modern biosensors [10]. By and large, it is considered as a milestone for all biosensing platforms that followed. Another important breakthrough came about a decade later from Bergveld with the invention of ion sensitive FETs (ISFETs) for neurophysiological measurement purposes [11]. This innovation is considered to have paved the way for the modern nanowire FETs, as well as many chemical sensors and PH sensors. As a consequence of these
IET Nanobiotechnol., 2013, Vol. 7, Iss. 1, pp. 721 doi: 10.1049/iet-nbt.2012.0005
developments, a big step was taken towards modern optical biosensing platforms with the application of SPR in a detector for the rst time in 1983 by Liedberg et al. [12]. The SPR was a physically well-understood phenomenon back then, yet its application to biosensing was a milestone for contemporary optical biosensors. On the other hand, mechanical biosensors mostly took after their MEMS counterparts which ourished with the growing popularity of MEMS in 1990s and 2000s. This review paper focuses on the current status and developments primarily in the past decade in different sensing mechanisms and corresponding biosensors used along with the levels of performance attained. These mechanisms have been categorised as electrical, mechanical and optical. Other emerging technologies are also presented. The vast majority of the biosensors covered in this study operate in a label-free manner as label-free detection is an important research eld in contemporary biosensors. In the next section, we introduce the concept of a biosensor together with the current technology trends and requirements that are essential in a thorough understanding of the topic. With the same line of reasoning, we dene the performance criteria that are used to evaluate the efcacy of different biosensor technologies. This discussion is followed by a detailed review of the most prominent sensing platforms being used or researched as a part of contemporary biosensor systems. Performance metrics for each type of biosensor are analysed whereas specic advantages and drawbacks are laid out with comparative benchmarks. Finally, based on an extensive literature survey, we also present promising new research directions
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& The Institution of Engineering and Technology 2013
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which are at their infancies but are expected to evolve into more mature elds of research in the near future with remarkable improvements on the current state-of-the-art.
Terminology and trends
Denition of the biosensor follows closely the broader denition of its chemical counterpart. A chemical sensor is a device that basically consists of a chemical recognition system and a transducer, which transforms chemical information into an analytically useful signal [13, 14]. Biosensors can be dened as such sensors with biological recognition elements and similar transducer structures [15]. Based on the signicant advancement of biosensors both in complexity and functionality, it is appropriate to include another element in the list of components, the interface to the outer world. As seen in Fig. 1, contemporary microand nanoscale biosensors can be broken down into three distinct components brought together, namely biomolecule recognition (sometimes called surface or capture), transducer and interface (or readout). Note that the conceptual schematic outlined in Fig. 1 is applicable to both labelled and label-free mechanisms. The solution that is being inspected for the desired biomolecules and its interaction with the biosensor surface constitute the biomolecule recognition or surface portion (sometimes it is called capture, as well). It is here that biomarkers or other biological entities of interest are captured whereas the surface acts inert towards other molecules in the solution. Furthermore, this part is closely related to surface science studies and contains the imperative step of biosensor surface functionalisation. The second component is the transducer which is generally characterised and categorised by the actual sensing mechanism implemented in the micro- or nanoscale regardless of the overall size of the detector. This sensing mechanism varies extensively among different biosensors such as electrical, mechanical, optical and nuclear magnetic. Finally, interface is the connection of the biosensor from the micro- or nanoscale to the external setup and includes the back end readout method for the sensor such as electrical or optical readouts, signal processing and amplication. The labelled and label-free detection methods differ in all three components outlined above. Labelled sensing can be considered as a comparatively indirect method that involves the use of intermediary uorescent molecules that bind to the analyte of interest and emit optical signals for detection which shapes the biomolecule recognition portion as well as the optical readout interface. On the other hand, label-free biosensors exhibit various recognition, transducer and readout techniques that are explained throughout this review paper and call for improvements in all of the three components. It can be said that there is a single method of detection in labelled mechanisms as opposed to label-free mechanisms which vary greatly in their way of sensing. As nanotechnology and related technologies progress, versatility in smaller scales also increases rapidly. To this end, the use of label-free methods employing nanotechnology opens up new challenges in sensing. One of the two important trends in biosensor development in this era is the development towards the implementation of label-free mechanisms. This kind of detection encompasses the vast majority of recent research in the eld of biosensors and forms the focus of this review. As mentioned above, labelled biosensing generally makes use
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Fig. 1 Schematic representation of the components in a typical biosensor utilising the widely used antibodyantigen interaction for sensing
The main components are biomolecule recognition (sometimes called surface or capture), transducer and interface (or readout). The biomolecule recognition component can be implemented using a variety of molecule pairs as mentioned. It should be noted that the transduction is not necessarily limited to one of the shown methods as well as the interface which can be more or less functional than illustrated. This portrayal and categorisation is valid for virtually all biosensors, and effectively outlines label-free sensing as well as labelled methods. The labelled methods can be more easily narrowed down compared with label-free methods in their denition because of the need to use specic chemistries for binding that emit light and optical methods for transduction (such as uorescence) and interface (specialised cameras, optical sensors etc.) parts of the sensor
of optical methods to detect stained or marked biological entities. On the other hand, label-free detection is basically dened as a biological sensing mechanism in which no
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staining, marking or any other sort of label attachment is required for operation. Thus the biological sample is virtually unaltered before and after the test. This sort of sensing eliminates the need for the binding of extra molecules which not only speeds up the process and preserves the originality of the specimen but also prevents any interference that the labelling might introduce in the binding site [16]. Another important factor that necessitates the improvement of label-free detectors is the trend towards in vivo sensing. Addition of other materials into the uidic system is understandably not desirable for such applications. The label-free biosensing trend is fueled by the advancing microtechnology and nanotechnology (along with smarter nanomaterials) as evidenced by the growing interest and expanding literature on these correlated topics especially in the last few years [17]. The other important trend in contemporary biosensors is the development towards integration with the readout component. In particular, integrated systems with complementary metal oxide semiconductor (CMOS)-based circuits attract growing interest. Also the interface component is developing towards electronic readouts as opposed to other methods such as integrated optics or labelled methods. The advantages of such an approach are three-fold. Firstly, monolithic integration with the readout results in more exibility on processing the transduced signal, thus, higher performance which is a major asset. Secondly, cost reduction and production in larger scales are enabled to meet the needs of the market, should the research be commercialised. Finally, throughput can be increased by multiplexing and more data can be extracted from the system by real-time sensing. Multiplexed sensing can be universally dened as the collection of data from multiple channels simultaneously. Such a detection scheme enables the capability of running multiple assays with the same sensor array and same sample at the same time, thus saving time. Real-time sensing and reaction monitoring is another feature that denotes that data can be collected continuously from the sensor as opposed to elapsed time sensing with two phase data collection (such as two separate steps of testing: sample immobilisation and then data acquisition). This way, concentrations of biological entities and characteristics of reactions taking place in the liquid can be monitored in real time as a function of time [18]. Multiplexing and real-time sensing are quite helpful assets attracting a lot of attention in todays biomedical research [17]. Putting together all of the advantages mentioned above, prove invaluable in reaching the ultimate goal of lab-on-a-chip (LOC) architectures and point-of-care devices that are easy to use while giving quick results and maintaining reliability. Many of the detection mechanisms naturally involve binding of the target biomolecule to a surface or a particle. This binding is generally required to be enhanced by using a coating, which promotes binding or adhesion. The type of molecule that is used for capturing the analyte molecule is sometimes referred to as the probe within this context. Sometimes multiple layers of linking molecules are utilised to increase the bonding strength. Biotinstreptavidin [19, 20], antibodyantigens [2123] and aptamers [24, 25] are among the most commonly used molecule pairs for surface functionalisation studies. Furthermore, using self-assembled-monolayers (SAMs) is becoming a popular choice in the recent literature [2628] as well as sandwich-type surface functionalisation and nanomaterials [17, 29]. In application, surface cleaning and preparation or
treatment are required before the functionalisation step. The choice of surface functionalisation method depends heavily on the type of sensing mechanism and the desired analyte.
Performance criteria
Well-dened gures of merit or performance metrics are essential to quantify the efciency of a sensor in a given real-life situation and to objectively compare its performance to different types of sensors. In the case of biosensors, sensitivity, limit of detection (LOD) and specicity are generally regarded as the most common criteria. Sensitivity is seen to be used in different contexts in the literature. Device sensitivity or output sensitivity is the response of the biosensor to the unit quantity of biomolecules applied to it. It can be visualised as the slope of the transfer function of the biosensor. This type of sensitivity is not stated as commonly as LOD or specicity in the literature due to non-linearities that arise from the complex nature of various transduction processes. Another context for the sensitivity is test sensitivity which has more to do with reliability. It can also be called test reliability and is generally dened as the ratio of the number of true positives yielded by the biosensor to the number of true and false positives in total. Test sensitivity can be considered as a measure of the reliability of the test with respect to a reference test, which should be rightfully assumed to yield correct results such as the enzyme-linked immunosorbent assay (ELISA) protocols. On the other hand, LOD, sometimes called detection sensitivity, is closely related to the device sensitivity denition. LOD can be dened as the minimum quantity of biomaterial that results in an output signal that is clearly discernible from the background noise. This measure of discernibility is generally a signal-to-noise ratio (SNR) greater than 2 or 3. The dimension of the biomaterial quantity can be given in mass units or moles if the molecular weight of the sample is well known. However, this might lead to ambiguities because of the delivery system. Most biomolecules are found inherently in a uidic environment, so the biosensor systems incorporate a microuidic channel or similar component to deliver the sample to the sensor component. Since the volume of the solution also comes into play, it is sometimes more reasonable and customary to account for it by also specifying the volume, thus using units of molar. If the extra complexity of including various microuidic quantities is not desired, units are given simply in mass, such as grams or daltons. The biomolecule recognition part of the biosensor plays an important role in the nal value of the LOD of the system. A specic parameter called the dissociation constant (Kd) is commonly used for biochemical reactions in this context, and it basically shows the afnity between the probe molecule and the target molecule to bind. For the general case, a smaller dissociation constant denotes better afnity between the probe and target. Although the exact relation between LOD and this parameter is somewhat complicated, it can be said that higher dissociation constants force an undesired increase in LOD for an otherwise identical biosensor. On a separate note, LOD is measured as a system-level parameter so it inherently includes the effect of the dissociation constant. This ultimately means that comparison between LOD parameters of different types of
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biosensors is more reasonable for biosensors employing similar probe and target pairs. There are various units used for sensitivity and LOD. Focus forming units (FFU) and colony forming units (CFU) are two examples to these different units used generally in imaging type biosensors corresponding to viable numbers of viruses and bacteria or fungus, respectively. Refractive index unit (RIU) is a common unit used in SPR type optical detectors biosensors because of the fundamental differences in its detection method. It refers to the change in refractive index of the media with respect to captured quantity of analyte. LOD can sometimes be specied in number of molecules or cells as well. Specicity is closely related to test sensitivity and dened as the afnity of the biosensor to respond to only the desired biomolecule, and no other specimen. Analyte-specic surface coatings are needed for achieving specicity in any given biosensor. If a biosensor responds to biomolecules other than the one that is desired to be captured, it has an increased chance of resulting in false positives, which undermines the reliability of the device to a great extent. Specicity does not have a common unit and is not inherently or even easily quantiable like LOD or sensitivity. Consequently, it is difcult to compare specicities from different studies as its denition varies slightly as well as the measurement technique. Most research studies demonstrate specicity by demonstrating the lack of sensory response to a small number of analytes (usually one or two) other than the one intended for detection. Although it is of utmost importance in the reliability of the overall sensor, specicity tests are usually conducted after other performance tests partly, because in most cases it is easier to modify the properties of the surface coating compared with redesigning the entire biosensor. Considering the medical needs of our time, point-of-care diagnostics require very low-LOD parameters and small sample volumes as the amount of available sample is scarce in most applications. For comparison of device performances with current methods employed in medical diagnostics, results from ELISA protocols can be used. For reference, ELISA generally provides LODs comparable to or better than roughly 1 pM for antibodies [30]. Signicant improvement in LOD is generally obtained through decreasing the scale, that is from larger scales to micro, and then from micro to nano. Further improvement is generally pursued in the transducer and interface aspects of the biosensors. On the other hand, real-life situations also demand a high specicity to prevent false positives. This fact calls for better understanding and more advanced engineering of the surface reactions through the use of nanotechnology with various chemical methods. biological modelling. For this reason, they are modelled as a complex network of capacitive and resistive elements which best ts the application [33]. Capturing mechanism varies in different studies and can be implemented by variants of macrolevel techniques to microlevel such as trapping by vacuum [34] as well as chemical immobilisation through surface functionalisation [23, 35, 36]. The use of out-of-plane electrodes is common as well as in-plane electrodes. There are examples of comb type in-plane electrodes being used for cell counting as in micro-Coulter counters and through cell lysate [37, 38]. Impedance spectroscopy is mostly used for performing measurements on captured whole cells. Measurements on tissues are generally not preferred as the heterogeneity in the ensemble can lead to incorrect results. However, heterogeneity has been exploited for sensing purposes as well. In a study by Han et al. [34], the developed sensor was able to distinguish between human breast tissue cell lines (MCF-10 A) and cancer cell lines (MCF-7, MDA-MB-231, and MDB-MB-435) through impedance measurements in the 100 Hz3 MHz range. This way, metastasized samples and different pathological states can be identied along with the progress of cancer in a given cell line. CMOS integration of electrochemical detection is important as it enables better ultimate signal quality, low cost, and multiplexing capability. Recently, the issue has been tackled by different research groups. Studies of Stagni et al. included on-chip capacitance measurements and analogue-to-digital conversion for detection of DNA hybridisation. This study presents improvements on signal quality and increased functionality using CMOS. A signal in the order of 1 nF was detectable for specic reactions at relatively low frequencies in the order of kHz [39]. Work by Levine et al. demonstrated the application of cyclic voltammetry in CMOS for measuring DNA hybridisation. A LOD of 400 strands/m2 was attained with 4 4 potentiostat electrode arrays [40]. On the other hand, Manickam et al. presented electrodes for electrochemical impedance spectroscopy in 10 10 arrays fully integrated on CMOS chips. Capable of operating in 10 Hz50 MHz range with 40 m 40 m pixels, the sensor provides a LOD value of 105 molecules for DNA detection. Protein detection with protein-G was also demonstrated for the rst time with integrated electronics [41]. The studies by Levine and Manickam demonstrate the multiplexing capability added to the system through CMOS integration. The capability of real-time reaction monitoring adds to the importance of these studies as well. Detection of small-sized biomolecules was also demonstrated by different groups, and results such as 3 nM for -hemolysin (HL) detection [42] and 10 pg/ml (equivalent to about 0.3 pM) for infectious salmon anaemia virus (ISAV) antigen detection [43] were obtained. EIS type of detectors has an advantage in terms of CMOS integration and providing extensive information as a result of measurement in a wide range of frequencies. Although detection of smaller molecules is also a common research subject in EIS, studies focus towards cell characterisation. The research on the subject is expected to progress towards characterisation of small biomolecules and increasing the relatively small signal levels produced by these detectors. Overall, the EIS method can be described as a characterisation method rather than a detection method in contrast to other electrical sensing platforms like nanowire FET biosensors [31].
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4.1
Sensing mechanisms
Electrical sensing
An electrical-sensing technique that has been used for a long time in biosensor systems is electrical impedance spectroscopy (EIS) [31, 32]. Such a biosensor basically consists of microuidics and capture electrodes. The analyte immobilised in some manner on an electrode or between the electrodes is electrically characterised for its impedance parameters at various frequencies. Components of a cell such as the lipid bilayer membrane or the cytoplasm are very complicated structures in terms of electrical and
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Another very popular method of electrical sensing has generally been associated with a structure that greatly resembles FETs. The FET biosensors take after their well-known electronic counterparts, but in this case the channel is replaced by a semiconductor structure coated with a specic receptor to which the target molecules are expected to bind. The principle of operation greatly resembles that of ISFETs. The electrical charges of bound molecules result in either accumulation or depletion of carriers, thereby changing the conductance of the channel and enabling biomolecular detection. For instance, for a p-type channel, the binding of a positively charged molecule decreases the conductivity, as it decreases the number of carriers contributing to current ow, whereas a negatively charged molecule increases conductivity. Fig. 2a shows a simplied schematic of the detection mechanism whereas Fig. 2b shows the scanning electron microscope (SEM) image of a ZnO nanowire FET biosensor [51]. The channel structure is occasionally a nanowire or sometimes a single walled carbon nanotube (SWCNT) [4446]. Bottomup and top-to-bottom type fabrication procedures are both valid approaches although the latter makes the integration more difcult. Nanowire FET biosensors are generally operated in either the back gate mode or the liquid gate mode. In the former, the gate voltage is applied to the substrate and it serves as the gate for FET [47]. In the latter, as the name suggests, the gate voltage is applied to the liquid that the FET biosensor is submerged in [48]. The most common channel material is silicon which is also the basis for very large scale integrated (VLSI) circuit and MEMS processing. Silicon can be easily patterned and doped with known methods; furthermore, integration with current fabrication technology is almost seamless [2]. The feasibility of using silicon nanowires for FET biosensors was demonstrated by Patolsky et al. by showing the versatility in detection of proteins, DNAs and single viruses. A LOD as low as 2 fM was attained for detection of prostate-specic antigen (PSA) biomarkers whereas maintaining an SNR greater than 3 [2]. A study, with a similar LOD result of 10 fM was carried out by Zhang et al. on reusable n-type silicon nanowires. In this study, covalent immobilisation technique was used to capture estrogen response element (ERE) dsDNAs [49]. Using nanowires in arrays introduces a statistical advantage for correcting non-uniformities along with a comparatively reproducible fabrication method. Consequently, Agarwal et al. demonstrated an array consisting of both p- and n-type silicon nanowire FET detectors. Biotinylated ssDNAs were successfully detected with an SNR greater than 6 at a level of 10 pM with n-type FETs [19]. One inherent drawback of silicon is the thin native oxide layer that covers its surface, which can result in impaired device performance. Therefore research in other materials, such as metal oxides is considered a worthwhile option [48]. Among these materials is ZnO, which is a high-bandgap material with high electron mobility that is widely used in biochemical applications. Liu et al. utilised ZnO nanowires for the detection of IgG protein in phosphate buffered saline (PBS) solution at a LOD of 50 ng/ml [50, 51]. Another metal oxide utilised for electrical biosensing was metal oxide chemical vapour deposition (MOCVD) coated IrO2 because of its chemical stability in pH changes and fast response time in solutions. Although, the premise of using conventional CMOS process is a major advantage, the device has only been tested in varying concentrations of PBS, and not yet tested with any biological specimen [52]. Ishikawa et al. used In2O3 in their sensor platforms to detect SARS virus N-proteins. A detection limit of about 1 nM was achieved with a 10 min long assay in 0.01 PBS [48]. Conducting polymers have also been proposed and used for cancer antigen CA-125 detection down to a limit of 1 U/ml by Bangar et al. A more recent study by the same group suggests an extremely low LOD of 0.1 fM for breast cancer gene ssDNA detection [53]. SWCNTs are also nding their way into biosensing as in many other elds of nanotechnology. Lo et al. demonstrated 10 ng/ml limit of detection for carcino embrionic antigen (CEA) tumor markers with Ni decorated SWCNTs [54]. There have also been studies on graphene-based devices. Ohno et al.s graphene based and
Fig. 2 Nanowire FET biosensors

a Schematic representation of the operation with binding of charged molecules b SEM image of a nanowire FET biosensor. The ZnO nanowire is placed on top of the Au electrodes using a focused ion beam (FIB) tool. The gure is reprinted from Ref. [51] IET Nanobiotechnol., 2013, Vol. 7, Iss. 1, pp. 721 doi: 10.1049/iet-nbt.2012.0005 11
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aptamer-modied nanowire FETs are reported to be capable of detecting down to about 0.3 nM of IgE in 100 nM bovine serum albumin (BSA) solution [24]. Even though promising results are shown to be possible with non-silicon nanowires, the major problem is their fabrication methods, which usually incorporate bottom-up growth. These methods are generally not compatible with CMOS fabrication. The majority of the nanowire FET biosensors rely on time domain measurements although there have been suggestions towards frequency domain applications. Such a study was carried out by Zheng et al. deriving from the fact that nanowire FETs exhibit icker (or 1/f ) noise characteristics without any or non-specic binding whereas immobilisation of the target analyte biomolecules results in a Lorentzian trend in the frequency spectrum. Detection by tting for this type of curves is claimed to improve LOD of given silicon nanowire FETs from 5 to 0.15 pM for PSA, without altering the actual structures [55]. Another method with frequency domain sensing was Mishra et al. which combines the reliable FET behaviour with EIS technique [56]. In this study, a thin protective layer of polyimide was used to prevent the exposure of polysilicon nanowire to the solution. A bacterial toxin, SEB, was successfully detected with a LOD of 10 fM in 1 PBS solution. Table 1 shows a comparison of the key results for the nanowire FETs from the literature. It can be inferred that silicon is the most common structural material with a high sensitivity. However, it is also seen that other types of nanowires are on par with and in some cases more efcient than silicon nanowires today even though their fabrication is limited by bottom-up methods. Whereas the advantages of silicon related to versatility in fabrication and mass production are apparent, they are readily challenged by the promise of high sensitivity of other types of nanowires that can be engineered to specic needs for better performance. The real breakthrough will probably be reached after discovery of more efcient integration methods of these nanowires with the electrodes. As a disadvantage, the nanowire FETs are compact biosensors with very small surface areas and it may take extended periods of time to capture the required amount of analyte molecules for a discernible output signal [57]. For this reason, very low-LOD values are expected to require long assay durations. However, there have been theoretical studies in the literature showing that electrokinetic effects such as electrophoretic force and electroosmotic ow could be exploited to reduce the assay time drastically [58]. 4.2 Mechanical sensing
Table 1
LOD
Comparison of key results from the literature about nanowire FET type biosensors Nanowire composition Si ZnO Si poly-Si In2O3 SWCNT Si Si Graphene Ni decorated Cond. Polymer Target analyte PSA IgG Biotinylated ssDNA SEB SARS CEA dsDNA PSA IgE ssDNA Ref. no. [2] [50] [59] [56] [48] [54] [49] [55] [24] [53]
2 fM 0.3 nM 10 pM 10 fM 1 nM 55 pM 10 fM 0.15 pM 0.3 nM 0.1 fM
Results in different units have been converted to molar units using commonly used molecular weights of corresponding analytes for reference 12
The subject of mechanical sensing has been researched extensively in various MEMS applications, the most famous examples being accelerometers and gyroscopes. For the past decade, sensors utilising MEMS technologies have been applied extensively to the biological domain as well. These sensors generally incorporate a cantilever structure. In essence, biomolecules immobilised to the cantilever cause a change in one of its mechanical characteristics such as the static deection or the resonance frequency of oscillation. In the former approach, the deection is caused by the change of the surface stress, whereas in the latter approach, the shift in the resonance characteristic is a result of the increased beam mass as biomolecules are captured as shown in Fig. 3. These two different mechanisms are addressed as static and resonant mode devices. Unlike electrical-sensing mechanisms with inherent electrical readout interfaces discussed in the previous section, mechanical biosensors commonly rely on optical setups for the readout. However, regardless of the readout, the performance of a mechanical sensor, especially a resonant mode sensor, varies greatly with the medium it is operating in. This degradation is related to the Q-factor of the system. The Q-factor of a system determines the quality of the output signal. In electromechanical systems, Q-factor of a resonating structure is a measure of the efciency of the oscillation and is dened as its peak resonance frequency divided by the frequency bandwidth. A higher Q-factor is desirable in the operation of a resonance mode mechanical biosensor as it translates to improved resonance characteristics, and therefore results in higher performance. Liquids, in general, cause deterioration by introducing additional drag force because of their viscous nature hence decreasing the Q-factor of the structure. For this reason, once the necessary amount of analyte molecules is captured, such biosensors might need to be desiccated before measurements are made. This operation points to a certain tradeoff between higher sensitivity and longer assay duration [60]. Static deection mode sensors provide a smaller amount of data to the experimenter for analysis. However, they compensate for it by the low LOD values they offer. Such a cantilever detector with optical readout was demonstrated by Backmann et al. in detection of the binding of AR-GCN4 antigens to scFv antibody fragments. With the help of fragmented probe antibodies and differential readings, a LOD of about 1 nM is obtained [22]. Similarly, Zhang et al. used multiple static mode cantilevers with reference detectors for differential readout. RNA immobilisation was achieved by hybridisation. In the end, a LOD of 10 pM was attained with ssDNA and oligonucleotide probes [61]. On the other hand, Shin et al. proposed a similar approach for dynamic mode devices. In their work, use of multiple resonant cantilevers was exploited to distinguish between the effects of surface stress and adsorbed mass through piezoelectric type electrical readout. Their resonant microcantilever sensors with the smallest one having a Q-factor of around 3000 were shown to detect 20 g/ml solutions of IgG proteins on biotinylated SAM surfaces and pinpoint the actual source of the resultant frequency shift owing to the differential readout
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number of process steps, increases throughput, and enables the real-time sensing applications. The Q-factor of the cantilevers was improved vastly by Ricciardi et al. who managed to obtain a value of 140 without electronic feedback by designing microplates rather than microbeams. Furthermore, the detection of 30 l Ang-1 mAb in a PBS solution of 25 g/ml was demonstrated [64]. Another structure that was proposed to increase the Q-factor was to use in-plane resonators. This type of cantilever has a smaller drag force acting on it, making Q-factors up to 249 possible. This way Tao et al. managed to fabricate silicon micromachined cantilevers with a detection limit of 2 103 CFU/ml was obtained for Escherichia coli samples [65]. Different materials and bimorphs in cantilever structures were proposed in the literature. A magnetostrictive alloy-based microcantilever with a measured Q-factor of 40 was demonstrated by Fu et al. for capturing Salmonella typhimurium. Two hours of assay time resulted in a 35 Hz shift in the sensors resonance peak which is at 6915 Hz in its rst mode in water. This kind of alloy brings the possibility of wireless actuation and sensing in a microcantilevers [66]. As opposed to majority of sensors that lie in the substrate plane and oscillate out-of-plane, an innovative vertical pillar structure, which is perpendicular to the substrate plane was recently proposed by Melli et al. [67]. This geometry helps to increase the adsorption rate signicantly by decreasing the required diffusion length for the analyte. Optical lever type readout was employed for DNA capturing with limits of detection of 1 M and 10 nM in less than 1 min and 2 h, respectively. Another interesting application worth noting is vibrating CNT-type biosensors. CNTs are highly sought after because of their superior mechanical properties and enhanced electrical characteristics, with respect to (and because of) their dimensions. In a study by Chowdhury et al., the idea of a SWCNT mass sensor is explored and it is claimed after simulations that 1024 kg of mass resolution should be possible with CNT-based nanobalances, although no measurement data are provided [68]. The most severe limitation of microcantilever type sensors is their signicantly degraded performance in a liquid environment. Although there were partially successful approaches involving electronic feedback for Q-control and in-plane resonators as covered, it was not until 2003 that a radically innovative way to overcome this limitation was proposed by Burg et al. in the form of suspended microchannel resonator (SMR) biosensors. As the name suggests, the suspended cantilever structure houses the microchannel in which the immobilisation occurs. The resultant microcantilevers are, therefore free of the liquid damping factor with very high Q-factors. This rst study yielded a surface mass resolution in the order of 10 ag/m2 over a bandwidth of about 4 Hz. The avidinbiotinylated BSA pair bindings were detected with optical readout [69]. Research on SMR by the same group yielded detectors with very high Q-factors in the order of 15 000. Goat anti-mouse IgG is detected with a LOD of 0.7 nM. Moreover, a new mass measurement method without immobilisation of the analyte on the inner surfaces of the SMR is proposed [70]. Studies of von Muhlen et al. on improved surface coating polymers along with reference microcantilever coupling yielded SMRs with LODs of 10 ng/ml for activated leukocyte cell adhesion molecule (ALCAM)-type cancer biomarkers [71]. Further studies on the subject brought about fully electronic readout with
Fig. 3 Principle of operation of static and resonant mode microcantilever biosensors

a Captured biomolecules change the surface stress of the microcantilever, causing it to bend b Immobilised biomolecules modulate the resonance frequency of an actuated, oscillating microcantilever. Both congurations can be read out by electrical and optical means
architecture [27]. Piezoelectric type signal generation and readout was also used by Lee et al. for PSA detection in cantilevers with composite structures and surfaces functionalised with SAMs. After running uorescence and specicity tests and comparing the results a LOD of 10 pg/ ml was reported [62]. Similar arrays of bimorph nanocantilevers were reported with a different readout method, namely, optical-thermo-mechanical excitation in the study proposed by Ilic et al. [63]. Only a very small circular area at the tip of the resonator was functionalised with gold coating. Detection of single dsDNAs was shown to be possible and a resultant mass LOD of 1.65 ag was reported whereas a 0.03 ng/l LOD was shown to be attained in the frequency response. The Q-factor after desiccation is given to be between 3000 and 5000 in this study. The improvement of Q-factor is a challenge in case the assay is desired to be carried out whereas the detector is immersed in the liquid. This type of assay decreases the
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piezoelectric sensing elements as was demonstrated by Lee et al. [72]. This study takes the concept one step forward as it enables multiplexed detectors and provides an ultimate mass resolution of 3.4 fg in a 1 kHz bandwidth with piezoelectric detection of polystyrene beads and budding yeast cells. The results were also shown to agree with those of optical lever method readout, ensuring the reliability of electronic readout. Another way to work around Q-factor limitations was proposed by Lu et al. as a resonator coupled with vertically directed and ordered nanowire arrays [73]. The nanowire array formed a photonic crystal (PC) which can detect DNA down to a LOD of 0.5 fM with quite linear output characteristics using optical interferometer readout. A highly improved mass per area sensitivity of 1.8 ng/m2 was demonstrated at the expense of cost efciency and simplicity of testing compared with other electronic readout sensors. Table 2 shows a comparison of the key results about mechanical biosensors from the literature. Although promising results are given in sensors with static deection, there is a lack of quantitative information gained from them along with a high dependence on ambient temperature, which makes it difcult to use piezoelectric readout methods, thus making CMOS integration and multiplexing harder to achieve. Even though there are concerns on their reproducibility, this drawback can be offset by use of differential readouts. Among the resonant mode cantilevers, there is a serious tradeoff between assay time and LOD, as the mechanical performance in liquid environments is signicantly hampered because of the Q-factor. Solutions such as in-plane resonators and electronic feedback have been proposed but they do not seem comparable to the promise of SMRs, which provide precision in the range of single molecule detection and additional advantages such as real-time reaction monitoring without the need for immobilisation. 4.3 Optical sensing light source, and an optical detector to collect the reected light as seen in Fig. 4. SPR sensors make use of surface plasmons which propagate along the substrate axis in the dielectric/metal interface. These surface plasmons interact with or can be coupled to the p-polarised component of the incident light [74]. The propagation vectors of these surface plasmons are sensitive to the change of the optical refractive index in the near-eld which is in turn sensitive to any change in the dielectric/metal interface where the biomolecules of interest are immobilised. Generally, the light reected from the back side of the functionalised surface is measured for one of three key quantities: the resonant intensity, wavelength or angle [75]. Ultimately, direct information related to the surface bindings of the biomolecules can be extracted. Recently, there has been a sharp increase in the number of publications on SPR sensors, and this type of sensor became a commercial choice. A reason for this is the wide range of applications for SPR sensors, ranging from explosive detection (trinitrotoluene) to gas sensing and biosensing [8, 76, 77]. Other reasons can be listed as the high sensitivity and real-time reaction monitoring capability. SPR technology seems to keep up with the trend followed by all biosensing platforms as it benets from the progress of
The eld of optical biosensing involves nanobiosensors as well as macroscale detectors. There exist a variety of methods including SPR sensors, PC, OMRRs and WGM detectors that have been used to this date. Here, we focus on SPR and PCs as they are the most common and the most recently developed and innovative approaches. By far, the most developed type of optical biosensors is SPR. A common setup consists of a dielectric lm coated with a thin layer of gold or silver and coupled to a prism, a
Fig. 4 Typical SPR setup based on reected light detection

Intensity and angle of the beam, after it exits the prism can be detected as well as the spectral response of the metal/dielectric interface (not shown in the gure). There are various SPR setups possible for different needs such as those that use transmission mode rather than reection mode or those that employ optical bres instead of prisms
Table 2 Comparison of key results from literature about mechanical biosensors

LOD 1 nM 0.3 pM 30 nM 10 pM 0.1 M 0.4 M 10 nM 0.7 nM 0.1 nM 3.4 fg 0.5 fM 2 103 CFU/ml Comments static deflection resonance mode resonance mode static deflection resonance mode resonating plates, in liquid resonating vertical pillars, in vacuum SMR SMR with reference and improved surface piezoelectric sensor SMR resonator coupled with PC nanowire array in-plane resonators, in liquid Q-factor N/A N/D 7800 N/A 3000 140 20 000 15 000 15 000 10 840 320 000 249 Target analyte AR-GCN4 PSA dsDNA RNA IgG Ang-1 DNA goat anti-mouse IgG ALCAM budding yeast cells DNA E. Coli Ref. no. [22] [62] [63] [61] [27] [64] [67] [70] [71] [72] [73] [65]
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nanotechnology. One of these studies by Wang et al. uses advanced surface functionalisation with aptamers and exploits the amplifying effect of gold nanoparticles to improve the LOD for adenosine down to 1 nM level [25]. In another recent study by Piliarik et al. involving nanorods, localised surface plasmons were imaged in polarisation contrast on a gold nanorod array. This setup was used to amplify the detection sensitivity by two orders of magnitude and detect 200 pM of short oligonucleotides [78]. Another such study on SPR sensing utilises low cost and easy to fabricate optic probes instead of prisms and a complicated optical setup. Sai et al. used U-bent optic probes and gold nanoparticles to attain a LOD of 0.8 nM for anti-IgG [79]. Although the technology is at a mature state, increasing the efciency by different designs is still sought after in the SPR domain. Recently, Zhang et al. completed extensive studies on coupling of bright dipolar and dark quadropolar modes of the SPR sensors. According to their studies, Fano resonances can be exploited by a careful geometrical design to improve performance [80]. On the other hand, studies are being conducted in extended versions of SPR method to further improve functionality. Such an extension is the Surface Plasmon Resonance Imaging (SPRI) technique which uses a charge-coupled device camera for the detector. A high throughput with low LODs and an improved ability to observe real-time reaction kinetics are possible with this method as well as the invaluable advantage of multiplexed detection. A study in this eld was done by Li et al. for detection of vascular endothelial growth factor (VEGF) in a biologically relevant concentration of 1 pM whereas a lower limit of 500 fM was reached during the detection of human thrombin. The use of aptamerisation in this study increases the sensitivity by almost four orders of magnitude [81]. Another variant of the SPR technique is the Surface Plasmon Fluorescence Spectroscopy (SPFS) which incorporates a similar setup with increased sensitivity. This technique makes use of uorophores excited in the metaldielectric liquid interface to provide an optical signal. Although it is a step away from label-free paradigm, it is claimed to provide a better sensitivity for measurements of direct binding interactions of low molecular weight molecules. In a study by Vareiro et al. probes functionalised with a SAM containing biotinylated and hydroxyl-terminated thiols were used to bind rst to streptavidin and then to anti--human chorionic gonadotrophin (hCG). A LOD of 0.6 pM was obtained for hCG [82]. PC detectors are a relatively new class of biosensors. They generally incorporate a periodic dielectric structure with a period that corresponds to a specic wavelength. This gives rise to a photonic bandgap which denes the part of the spectrum that is not allowed to propagate through the structure, that is reected. In a PC biosensor, generally a defect is introduced into the device on purpose so that a shift in the peak wavelength value (PWV) is observed in the reection or transmission characteristics. Both the periodic structure and the dielectric constants are instrumental in shaping the reection or transmission spectra and changes in either one can be attributed to changes in the surroundings of the defect. This way, detection of biomolecules is possible through microcavities. Another valid method is to implement waveguides in the lattice structure. An example of PC biosensors in which the transmission spectrum is observed was given by Lee et al. with a
minimum detectable protein mass of 2.5 fg [20]. Silicon-on-insulator (SOI)-type substrates were etched with periodic cavities of 270 nm diametre with a single defect of 140 nm diametre in the centre to observe the spectrum from a tunable laser source. After thermal and chemical treatments, application of glutaraldehyde enabled the detection of BSA. Specicity was also demonstrated by using streptavidinbiotin binding method. Fig. 5 shows an SEM image of this PC biosensor [20]. Skivesen et al. used a similar approach in their detector, employing a blank line, which acts as a waveguide in the propagation direction instead of a single microcavity defect in the PC. The cut-off wavelength of the waveguide in SOI with cavities was used for detection of BSA down to a LOD of 0.15 M [83]. Reection spectrum is also used for detection in some cases. Such a biosensor was demonstrated by Block et al. on exible plastic substrates to improve robustness and reliability [84]. Lactoferrin of 12.5 g/ml was detected by biotinylated heparin probes after surface treatment. The structures were exible up to a radius of curvature of 15 cm, which paves the way for more robust PC biosensors. Highly specic detection of specimen such as rotavirus was demonstrated by Pineda et al. with the potential for large quantity detection using PWV shifts in environmental water supplies at a LOD of around 36 FFU [85]. Real-time detection has also been investigated. In such a study, PC biosensors fabricated with UV curable polymer sheet gratings and high refractive index TiO2 lms have been used as sensor arrays. Chan et al. demonstrated imaging of cells cultures using the reected spectrum approach to detect apoptosis or proliferation of a type of human breast cancer cells, MFC-7, upon application of various plant extracts [86]. This study shows the potential of real-time monitoring of the specimen and is claimed to be an alternative to SPR imaging for the same purposes. Another study on real-time monitoring deals especially with the detection of low-mass interactions through the use of total internal reection geometry (TIR) [87]. Proteins with molecular weights as low as 244 Da have been shown to be detected. Ultimately, 1 nM biotin-20T solution was detected with an SNR up to 2000. Nanostructured surfaces are expected to further improve this performance. Addressing
Fig. 5 Two-dimensional PC microcavity biosensor proposed by Lee et al.

Figure is reprinted from Ref. [20] 15
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the nanostructured surface issue, surface area enhancement of PC biosensors coated with TiO2 lms has been demonstrated by Zhang et al. [88]. The surface area is increased up to four times by use of glancing angle e-beam deposition which forms nanorod structures on the surface. The sensitivity is shown to be increased rather indirectly as this approach enables more material to be adsorbed on the effectively increased surface area. Table 3 provides a comparison for the optical sensors reported in this section. The widely used and commercialised SPRs provide low LOD values with real-time reaction monitoring capability. Owing to the almost saturated state of inherent sensor performance, new approaches to the subject are necessary such as improved surface functionalisation and advanced optical setups. On the other hand, SPR performance seems to be rivaled quite readily by PC type detectors. The PC type detectors have been shown to be usable for reaction monitoring as well, and furthermore have the size and scale advantage in detection of small or low surface coverage molecules [87]. 4.4 Other sensing platforms Biosensing is performed by binding of these magnetic nanoparticles to the target analyte. Generally, bigger particles or, clusters of particles as opposed to homogeneously distributed particles result in longer relaxation times and in turn, higher output signal [89, 90]. There has been interest in miniaturising NMR systems especially because of cost issues, rst to less claustrophobic systems with open sides and smaller form factors, then to desktop-sized sample processing systems, and nally to handheld devices [91]. Although there were studies on implementing the important individual components with microfabrication technologies [92, 93], a study that resulted in fully miniaturised sensors was carried out by Lee et al. and yielded a portable NMR sensor based on the above-mentioned magnetic relaxation mechanism which was capable of detecting avidin down to 3 nM LOD using cross-linked iron oxide. A more impressive LOD of 1 pM is claimed to be reached using magnetic microparticles for mouse IgG detection [94]. Fig. 6a shows the schematic for the proposed NMR biosensor whereas Fig. 6b illustrates a SEM image of the magnetic nanoparticles used for NMR studies [94, 96]. There also have been other and further miniaturisation efforts on this subject. For instance, CMOS type coils were employed in addition to the RF integrated circuit for a sensor developed by Sun et al. [95]. Another study by the same group, Lee et al. integrated embedded microcoils with a microuidic channel to increase SNR to quadruple the values given in previous studies [96]. Another emerging biosensing platform is that of nanopores, rst demonstrated by Kasianowicz et al. which can be thought of as a nanoscale Coulter counter [100]. A nanopore biosensor generally consists of a well-controlled microuidic channel and a small conned space called a nanopore in which electrical measurements can be made primarily on DNA or RNA strands, proteins and protein complexes [97, 98]. The microuidic portion serves to move the strands with electrophoretic force through the nanopore. Among the most common materials for nanopore fabrication are proteins such as HL [99103] and solid-state materials such as silicon and silicon-based insulator lms such as silicon nitride and silicon oxide and graphene [104108]. Fig. 7a shows a SEM image of a nanopore in silicon oxide [108]. Other possibilities are also explored, such as functionalising nanopore surfaces with complementary DNA for specic sequence detection [109] and the use of CNTs [110]. One important gure of merit for nanopore sensors that is not common among other types of biosensors is translocation velocity. This parameter is determined by many factors such as the size of the molecules, that is the sequence length in DNA-like strands, transport medium, nanopore diameter and surface and membrane optimisation. The best (lowest) translocation velocities reported are in the range of 0.1 nucleotides/ms for HL-type nanopores and 100 nucleotides/ms for solid-state nanopores [111]. In basic mode of operation, a constant voltage is applied on the nanopore and the current is read out. As molecules are translocated through the cavity electrophoretically, they change the conductance within the cavity resulting in a change in current which is dependent on the type of the nucleotide. Fig. 7b shows the schematic representation of sensor operation. This is called tunneling current mode whereas sensors using capacitance measurements are also seen in literature [97, 112]. Long chains of DNAs and RNAs can be identied in a label-free manner with
There are other promising types of biosensors that can be considered outside the three basic archetypes discussed in the previous sections. This category includes Micro Nuclear Magnetic Resonance (NMR), nanopore and surface acoustic wave (SAW) biosensor platforms. An emerging type of biosensors that has garnered interest recently is the NMR. NMR biosensors make use of a principle somewhat similar to that of magnetic resonance imaging (MRI), which went on to become one of the most versatile medical diagnostic tools. The work in the literature about NMR generally includes the usage of a permanent magnet and microcoils for transducing, RF circuits for readout, and magnetic nanoparticles in a microuidic channel for sensing and delivery. The nanoparticles placed in the solution have paramagnetic properties and this causes a magnetic dipole to be created in the region under the external magnetic eld. This irregularity gives rise to a magnetic eld gradient and disturbs the nuclear spins of the protons in the nearby water molecules. This disturbance is read out as a magnetic eld resonance signal by shortening of the longitudinal or transverse relaxation times [89].
Table 3
LOD
Comparison of key results from literature about SPR and PC type optical biosensors Comments Target analyte Adenosine anti-IgG DNA thrombin hCG BSA lactoferrin biotin-20T BSA rotavirus Ref. no. [25] [79] [78] [81] [82] [83] [84] [87] [20] [85]
1 nM 0.8 nM 0.2 nM 0.5 pM 0.6 pM 0.15 M 0.15 M 1 nM 19 fM 36 FFU
SPR, aptamer based Au nanoparticles SPR with U-bent probe Localised SPR with Au nanorods SPRI with aptamerisation SPFS PC with waveguide PC on flexible substrate PC with TIR geometry PC with single microcavity PC, large qty. detection
Results in different units have been converted to molar units when possible using commonly used molecular weights of corresponding analytes for reference 16
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Fig. 6 Portable NMR sensors

a NMR sensor proposed by Lee et al. consisting of a microcoil array, microuidic delivery and signal processing RF IC in a single, portable, hand-held device b Mn-doped ferrite magnetic nanoparticles. The gures are reprinted from Refs. [94, 96]
low-cost nanopore sensors. Advanced nanopore detectors are envisioned to eliminate the need to amplify the number of strands using polymerase chain reaction (PCR) techniques or the use of other extensive protocols prior to the actual measurement [111]. Recently, coupling of nanowire technology with nanopore devices has been demonstrated by Xie et al. [113]. The nal type of biosensor to be considered in this section is SAW detectors. A SAW is basically a high-frequency (30 500 MHz) acoustic wave that travels along the surface of the substrate and is sensitive to any modications on its path of propagation. Generally, higher frequencies result in better sensitivities but also require thinner substrates which do not
usually provide mechanical reliability [114]. The most common method to generate and sense SAWs is the use of piezoelectric materials. After the wave is generated on the actuator side by interdigitated transducer (IDT)-type electrodes, necessary information on the desired quantity, such as surface bindings in the case of a biosensor, is extracted from the measured frequency or phase at the sensing side again with an IDT. Lately, there have been innovative approaches such as incorporation with SPR-type sensors to increase functionality [115] and using circular structures for elimination of the diffraction of generated SAWs in CMOS technology (see Fig. 8) [116]. Although, the SAW
Fig. 7 Nanopore biosensors

a SEM image of a silicon dioxide-based nanopore. The SEM image is reprinted from Ref. [108] b Schematic representation of a nanopore sensor. The voltage applied to the solution forces an ssDNA to move through the nanopore. Meanwhile, the current ow is monitored under a constant voltage on the nanopore. The current level changes according to the type of nucleotide passing through IET Nanobiotechnol., 2013, Vol. 7, Iss. 1, pp. 721 doi: 10.1049/iet-nbt.2012.0005 17
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difcult to compare different studies. Electrical impedance analysis systems generally suffer from low-signal levels compared to other transduction techniques. This is the major reason why integrating them with CMOS technologies is a gateway to major improvement in this eld. It also alleviates the need for extensive test setup and equipment. Similarly, nanowire FET-based sensors are inherently very easy to integrate with the CMOS readout circuits. This would allow them to be less costly while drawing more modest amounts of power. They have no movable parts and their form factor is small, making them more reliable in the long run. The issue of a possibly long assay time is still present but it was proposed that this problem can be tackled using approaches such as electrokinetic effects. Nanowire FET type sensors fully exploit the advantages of nanotechnology and generally exhibit very low LODs. Microcantilever-based mechanical sensors yield high performance that scales inversely with size, that is smaller the detector, better the sensitivity. Also fast response times are possible with most mechanical sensors. However, static mode sensors have integration and multiplexing problems whereas the resonant mode sensors operate with hampered performance in uidic environments. Furthermore, resonant mode sensors with optical readouts have comparably limited dynamic ranges. Both performance and versatility of mechanical type biosensors have been improved substantially by SMRs. Recently, other technologies such as PC nanowires found applications as hybrid mechanical biosensors and exhibited great potential. Sacricing simplicity of measurement and cost efciency at some level, this biosensor exhibits the best DNA LOD performance in terms of Molar units reported up to this date to the best of our knowledge. SPR biosensors have been extensively researched and commercialised because of their reliability, capability of measuring the reaction kinetics and real-time monitoring capability. Yet, high-performance sensors require high-quality optical setups. This results in rather costly and less portable systems. PC-based detectors are still at a comparatively early stage of research but show great promise in terms of sensitivity. Unfortunately, they also require high-quality lasers, polarisers, and other optical setups to function. NMR biosensors require high-performance RF electronics especially with large magnetic particle sizes but are compliant with CMOS RF process ows, and offer high sensitivities
Fig. 8 SEM image from the circular SAW devices proposed by Tigli et al. with circular IDT electrodes to minimise diffraction losses
Figure is reprinted from Ref. [116]
biosensors are sensitive and provide real-time measurement capability, they have an inherently degraded performance in liquid environment which has to be carefully accounted for.
Conclusions and future outlook
Biosensors have been improving in performance and versatility at a fast pace. Advances in nanotechnology, MEMS, NEMS and other very small-scale fabrication technologies help the biosensors progress even further and even faster. The eld is expanding at a fast pace with emerging innovative technologies, such as NMR imaging, nanopore sensors and even memristor-based new approaches. Table 4 provides a quick summary of the archetypes of sensors discussed in this review paper. It should be noted again that while LOD is a good metric to compare different sensors, it does not tell the whole story: assay time, type of analyte, specics of surface chemistry (therefore the specicity), readout technique, and quality of signal processing are just a few of the many parameters that are instrumental in the overall quality of the biosensor. Among these other parameters, specicity is not discussed in the table on purpose as its quantication is not normalised as in the case of LOD, and it is measured or demonstrated in many different ways which makes it very
Table 4 Summary of the categories of biosensors discussed in this review

Type Best LODs reported 0.3 pM 1 fM 0.5 fM 0.5 pM 19 fM 1 pM N/A N/A Typical types of analytes Estimated relative cost low to average low low to high average high low low low Readout interface Commercial products yes no no yes no no no no
EIS nanowire FET mechanical SPR PC NMR nanopore SAW
cells, proteins proteins, DNA, viruses proteins, DNA/RNA, bacteria, viruses proteins, viruses proteins, viruses proteins DNA/RNA proteins
electronic electronic electronic/optical/mass spectrum optical optical electronic electronic/optical electronic
All listed types of biosensors have been shown to be capable of label-free detection. Owing to the difference of measurements and unit dimensions, respectively, LOD parameters for nanopore and SAW type sensors are not included in the table. Specificity is not included in the overall comparison for two reasons: because of the inability to come up with a solid metric for quantifying specificity for different types of analytes and due to the variety of the ways it is seen to be demonstrated in different studies 18 IET Nanobiotechnol., 2013, Vol. 7, Iss. 1, pp. 721 doi: 10.1049/iet-nbt.2012.0005
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along with high test sensitivities, meaning low false-positive ratios. This technology is currently at the brink of commercialisation, provided that the overall miniaturisation efforts and integration with CMOS yield competitive performance in real-life medical diagnosis situations. Nanopore-type biosensors combine the ability to examine single nucleotides in a given DNA or RNA sequence. Currently, combining solid-state nanopores with biological probes to create hybrid nanopore sensors, increasing the specicity of the sensors, and decreasing the translocation velocity seem to be the biggest challenges in this eld. SAW-type sensors are sensitive yet possess limited functionality in liquid environments, which undermine their throughput. Realisation of CMOS readout integrated SAW biosensors are demonstrated as proof-of-concept but their mass production and full integration is not yet accomplished. It is the widely adopted opinion that the biosensing platforms in the micro- and nanoscale developed so far lend themselves to being smart alternatives to labelled techniques. This is partly because of the envisioned development towards in vivo sensing and partly because of the need for more rapid assays with decreased durations that yield more quantitative data. On a separate note, almost regardless of the sensing mechanism, integration with monolithic readout circuits adds the invaluable advantage of multiplexing, decreases cost, increases portability, improves signal quality and device functionality, and enables options in mass production. This applies best to biosensors that can be fabricated using standard CMOS technologies and those with electronic readouts. Analysing the current state-of-the-art, our predictions suggest the future of biosensors lies in the integration of portable and label-free systems benetting from the full array of advantages of the nanotechnology toolset with high-functionality electronic readout circuits. With the current pace of advancements in the eld, we expect such point-of-care devices to be highly commercialised after the next decade, which will be lled with many exciting results in the eld of biosensor research.
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Acknowledgments
The authors would like to thank all members of the BioNano Research Team of University of Miami, Department of Electrical and Computer Engineering, and Department of Pathology for their support. They would also like to acknowledge Mr. Marcos Feddersen for his help with the nanowire gure.
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Biosensors in the small scale: methods and technology trends

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Terminology and trends

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Fig. 2 Nanowire FET biosensors

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2 fM 0.3 nM 10 pM 10 fM 1 nM 55 pM 10 fM 0.15 pM 0.3 nM 0.1 fM

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Fig. 3 Principle of operation of static and resonant mode microcantilever biosensors

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Fig. 4 Typical SPR setup based on reected light detection

Table 2 Comparison of key results from literature about mechanical biosensors

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Fig. 5 Two-dimensional PC microcavity biosensor proposed by Lee et al.

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1 nM 0.8 nM 0.2 nM 0.5 pM 0.6 pM 0.15 M 0.15 M 1 nM 19 fM 36 FFU

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Fig. 6 Portable NMR sensors

Fig. 7 Nanopore biosensors

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Conclusions and future outlook

Table 4 Summary of the categories of biosensors discussed in this review

EIS nanowire FET mechanical SPR PC NMR nanopore SAW

electronic electronic electronic/optical/mass spectrum optical optical electronic electronic/optical electronic

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IET Nanobiotechnol., 2013, Vol. 7, Iss. 1, pp. 721 doi: 10.1049/iet-nbt.2012.0005

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IET Nanobiotechnol., 2013, Vol. 7, Iss. 1, pp. 721 doi: 10.1049/iet-nbt.2012.0005

& The Institution of Engineering and Technology 2013

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