Design and development of advanced biosensing systems for the rapid detection of antibiotics

ABSTRACT The indiscriminate use of antibiotics has led to antimicrobial resistance (AMR) that is affecting both public and animal health, worldwide. To circumvent this and prevent antibiotics from entering the food chain, it is essential to detect them rapidly at the source. As most of the conventional methods used for the antibiotic detection in biological, food and environmental samples are time consuming and expensive, a suitable alternative is served by the biosensors which provide rapid, cost-effective and user-friendly platform for portable sensing applications. Development of devices using lab-on-a-chip technology has potential application in the field of development of biosensor-based antibiotic detection kits. This review gives a detailed discussion of various biosensors developed for antibiotic detection. Recent techniques such as the integration of biosensing and microfluidic technologies, along with the simulation softwares employed which aids in the miniaturisation of the biosensing devices are also highlighted in this review.


Introduction
According to the World Health Organisation (WHO) reports, antimicrobial resistance (AMR) has been viewed as one of the severe global threats, which leads to the rise of many drug-resistant diseases in the near future [1]. The main cause is the increased misuse of antibiotics which are very commonly used against bacterial infections; they kill the infectious bacteria and not the mutant ones that are resistive to the drug. These drugs do not get completely metabolised in human or animal body and are excreted into the environment and enter the food cycle through animals and environment [2]. Apart from this, the antibiotics can indirectly reach humans through meat and dairy products, as they are extensively used in veterinary medicine. An effective system for the monitoring of antibiotic drug use is thus essential.
Antibiotics are mainly classified based on their site and mechanism of action in the biological system. Some target cell wall synthesis, some on nucleic acid synthesis and some target the protein synthesis [3]. Based on the chemical structures, antibiotics are classified as aminoglycosides, sulphonamides, tetracyclines, β-lactams, quinolones, CONTACT Renu Vyas renu.vyas@mituniversity.edu.in This article has been republished with minor changes. These changes do not impact the academic content of the article.
Supplemental data for this article can be accessed here. macrolides and fenicols [4]. The antibiotics cause some toxic side effects on human body such as ototoxicity, nephrotoxicity, hepatotoxicity, decrease in metabolic rate, anaemia, allergic reaction, hypersensitivity etc. in living beings [5][6][7]. The different antibiotics and their adverse effects on human health are depicted in Figure 1. A few others such as sulphonamides and trimethoprim cause blockage in metabolic pathways [8]. Therefore, WHO is encouraging all nations to keep monitoring the spread of AMR and find ways for mitigation. Some of the mitigation methods include monitoring of antibiotic prescribing, promoting rational use of antimicrobial agents introducing new treatment recommendations, educating health centre staff on risk and benefits of antibiotics etc [9]. It is very important to monitor the wide use of antibiotics in environmental samples such as air, water and food samples [10]. This review covers literature on the research work conducted in different technologies employed in antibiotic monitoring in the last five years. It encompasses the conventional methods as well as latest ones such as biosensors. Recent approaches in miniaturisation of detecting device using biosensors or nanosensors involve lab-on-a-chip technology by integrating microvalves and micropumps. This review has provided a brief overview of the lab-on-chip technology integrated with biosensors in antibiotic sensing applications (Table S1) [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28]. The materials used in fabricating lab-on-a-chip models and the simulation softwares aids in characterisation of the same are also discussed in this review.

Antibiotic detection techniques
According to the chemical composition of antibiotics, many chemical sensors and biosensors are employed in identification and detection of specific antibiotics [29]. Chemical assays, microbiological assays and immunoassays are mostly employed in the analysis of the concentration of antibiotic present in any matrix. Microbiological assays have a lot of limitations as they require laboratory facilities along with expertise for analysis that limits the technique for on-site use. Considering this fact, chemical assays are more suitable in portable equipment. They are more precise, cost-effective and less time consuming. However, it is mandatory to undergo preprocessing of the environmental water samples to enhance the antibiotic among other toxic elements. In on-site monitoring of antibiotics present in environmental or food sample, preprocessing is required for avoiding external interferences. The pretreatment techniques vary depending upon the type of assay and diverse matrices. Filtration is one of the common pretreatment methods used worldwide. A study on effect of membrane filtration carried out in detection of 28 antibiotics along with 7 NSAIDs proved that glass fibre filter with 0.7 µm thickness showed better performance than nylon filters and mixed cellulose ester filters on all the targets present in surface water samples [30]. The use of suitable surrogates prior to filtration and acidification after filtration was also suggested for different classes to avoid losses due to adsorption. Solid Phase Extraction (SPE) is another commonly applied pretreatment method for liquid samples that is more efficient and employed in different matrices according to the target analytes. But this is a lengthy procedure and time consuming [31]. SPE was used along with centrifugation in the detection of sulphonamides for interference-free detection with good accuracy [32]. Ultrasonic extraction followed by centrifugation is very often applied in pretreatment techniques that require well equipped laboratory facilities. Recently, Ye Jiang and team had developed a device to be used for on-site detection of toxic chemicals integrating pretreatment technologies such as ultrasonication, centrifugation and ultrafiltration in a single unit [33]. Though device takes 11 min for the entire preprocessing of sample, the multiple sample transfer for each step can be avoided and high throughput can be achieved. Liquid-liquid extraction (LLE), solid phase micro extraction (SPME), microwave-assisted extraction (MAE), supercritical fluid extraction (SFE) are another few methods employed in pretreatment of environmental samples [34]. Table S2 highlights the commonly used sample pretreatment techniques in antibiotic detection in diversified matrices [32,[35][36][37][38][39].
Purification and pretreatment of the sample before subjecting to detection requires equipped laboratory techniques and involves challenging procedures such as chromatography, spectrometry etc. Immunoassays, on the other hand, provide the most sensitive technique in antibiotic detection, based on antigen-antibody reaction and hence it is a very specific and a promising approach while using along with biosensors. Traditionally employed technologies for antibiotic detection in any of these assays include chromatographic techniques, ELISA, electrophoresis, which are briefly mentioned in the following sections.

Conventional methods in antibiotic detection
Chromatography, electrophoresis, ELISA are the conventional methods of antibiotic detection [40]. Chromatography techniques are widely accepted technique in the detection of antibiotics in various biological matrices due to their versatility and ability to separate the drug molecules from a compound mixture [41]. They provide more reliable and sensitive analysis using a very small sample volume, as compared to any other analytical techniques. According to the application, several chromatographic techniques are available namely Paper Chromatography, Column Chromatography, Thin Layer Chromatography, Gas Chromatography, Liquid Chromatography and High Performance Liquid Chromatography [42,43]. High Performance Liquid Chromatography (HPLC), involving principles of column chromatography, is one of the powerful analytical tools in determining concentration of antibiotics such as tetracycline [44], aminoglycosides [45] etc. In a study, eight types of aminoglycosides were determined simultaneously from animal feeds using HPLC, in which linear calibration curves were obtained indicating the high sensitivity of the method [46]. Analysing the aminoglycoside residues in foodstuffs and water bodies is really challenging in developing countries as the impurity profiling and extraction is difficult. Fast and economic methods like microbial assay can be used for screening to overcome this challenge, but they are relatively inaccurate and non-specific [47]. Photodiode array is often used along with HPLC so that absorption spectra of samples can be recorded for a wide range of wavelengths, which gives qualitative information about the samples [48]. β-lactam and fluoroquinolone were detected and separated from pharmaceutical formulations and biological fluids using a more selective and sensitive technology, Thin layer Chromatography (TLC). This method proved to be very convenient and economical by using silica gel as adsorbent for the separation of the two drugs [49].
Electrophoresis is another separation technique in which charged particles or molecules migrate according to the electric field provided. A high voltage must be supplied to ensure the electroosmotic and electrophoretic mobility. Esteves and team have screened six antibiotics simultaneously from milk samples [50]. The retention time and migration time were observed to be less in capillary electrophoresis as compared to HPLC by studying the effect of pH, temperature, voltage and other factors affecting mobility. Even though these techniques give better results, they are expensive, time consuming, laborious and tedious. The widely used techniques that are used recently in the field of antibiotic sensing are lab on chip and mass spectrometry. Despite the fact that mass spectrometry results are selective and sensitive, the instrumentation required is costly and are prone to interference effects. In comparison with this, lab-on-chip technology has more operational efficiency involving less number of fluid volumes and is the better option for simultaneous analysis of multiple assays [51]. Emerging technologies using biosensor-based detection have paved a way in detection of antibiotics with less time and cost.

Biosensing principles for antibiotic detection
The advent of biosensors has brought a remarkable advance in providing a sensitive, miniaturised, low cost sensing technology. The biosensors involve mainly three elementsbiorecognition element (enzyme, DNA, cell receptors or antigen etc.), the transduction part such as electrochemical, piezo electric, optical sensing etc. and an electronic system including signal amplifier, processor and display ( Figure 2). The biorecognition element forms affinity pair with the target in sample and the physical changes due to this is sensed and converted into electrical signals with the help of transducers. The main advantage of using biosensors is that it overcomes the limitations of the above-mentioned conventional methods such as long detection time, time-based human intervention and usage of reagents in large quantities [52]. Commonly used bio-transduction principles employed in antibiotic detection are highlighted below.

Electrochemical biosensors
Electrochemical biosensors consist of biosensing elements coated on the working electrode, that are used to detect and quantify the target molecule using a simple design to obtain direct electronic readout [53]. After binding of the target molecule with the biosensing element, electrical signal is generated according to the chemical change that occurred in the working electrode ( Figure S3(a)). These biosensors are also known to provide an excellent limit of detection (LOD). The special feature enabling their wide use in application areas are portability and low cost. Due to these reasons, they have been considerably used in environmental monitoring, disease diagnosis, food analysis and other biomedical applications [54][55][56][57]. According to the measured parameters, electrochemical biosensors can be classified as amperometry and voltammetry techniques based on the mass transfer rate to electrode surface and difference in the potential between working and reference electrodes, respectively [58,59].
One of the widespread technologies in fabrication of electrochemical sensors is the use of screen-printing technology ( Figure S3(b)). Screen printed electrodes are cheap, can be bulk fabricated and are disposable ones too. The working electrode used in this technology is either coated with ink, commonly Ag/AgCl or carbon due to its inert nature [60]. This technology was used for detecting different classes of antibiotics in water in very low detection limits. Electrochemical sensors using Screen printed electrode (SPE) technology were used in analysis of many drug molecules present in blood, diary and other food samples [61]. Carbon nanotubes screen-printed sensors for gentamycin sulphate detection showed LOD of 75 nM [62], sulfamethoxazole detection using amperometry yielded LOD of 22.6 ± 2.1 µM [63]. Aptasensors are another class of biosensors, that use single stranded DNA (ssDNA) as the biorecognition unit. DNA aptamer-based sensor showed sensitive results in Tetracycline detection with an LOD of 10 nM [64]. In the detection of ciprofloxacin in spiked water samples, differential pulse voltammetry (DPV) was employed where aptamers were used along with electrochemical detection [65].
Further miniaturisation of the device with fast response can be carried out using nano particle-based electrochemical biosensors. In a recent study, a gold nanoparticle/carboncoated thread-based wearable electrochemical sensor was developed for the quantification of uric acid in human metabolite fluids [66]. In yet another study, GO/ZnO nanocomposite coated glassy carbon electrode (GCE)-based sensor was reported with high selectivity, stability, reproducibility and low detection limit resulting in efficient determination of chloramphenicol in liquid samples [11]. Another rapid, highly sensitive, labelfree sensor was recently used in identification of antibiotics in meat sample with a detection limit of 10 ng/mL [12], wherein biosensor fabrication was made possible by the vapour deposition of electrodes with gold particles on a Polyethylene terephthalate (PET) substrate. Although characterisation of electrochemical biosensors requires more sophisticated laboratory instruments, yet it is one of the popular techniques used in miniaturised and portable biosensor-based devices.

Optical biosensors
Apart from electrochemical biosensors, optical biosensors are another low cost alternative for sensing antibiotic drug residues. In optical biosensors, the biosensing element is integrated with an optic transducer system and the measurements are taken mostly by photometric/colorimetric methods. The instrumentation involves either the measurement of absorbance as the measure of quantification of target molecule or the measurement of light output by a luminescent process. This technique involves a light source, whose wavelength matches with the absorption spectra, a light detector and a channel for the light to travel [67,68] and in order to minimise transmission loss, optic fibre media is employed ( Figure S4(a)). As the optical method provides a sensitive approach among all biosensors, it is a better option for antibiotic detection in food and environmental samples [69]. Colorimetric techniques have an advantage of better visualisation of results with high sensitivity, hence the optical system provides better results suitable for the identification and quantification of any substrate in sample.
In biosensor applications, mostly intrinsic optical sensing methods are used, which are more sensitive as they involve direct measurement of change in optical properties of transmitted light. One such method is Surface Plasmon Resonance (SPR) technique, in which biosensing film is coated at the interface between two media of dissimilar refractive indices [70] (Figure S4(b)). Hence this technique is very sensitive and is used in most colorimetric assays, as the resonant wavelength varies according to the change in refractive index at the surface of coating. Application of SPR technique was also achieved by removing the cladding of optic fibre up to a distance and coating the biosensing element on the cladding removed portion. This technique has a large potential in rapid detection of any bioanalyte in environmental and biomedical applications [71]. The fibre can be modified in any configuration as bended or tapered portion gets exposed to the analyte [72]. Several studies have proved that electrochemical and optical techniques are more powerful in detection and real-time analysis of tetracyclines [64], quinolones [59] etc. Mason and team reported a solution of sensor fusion for detection of antibiotics in aqueous solutions [73]. They had experimented different sensing methodologies involving capacitive sensors, optical sensors and microwave sensors for monitoring the concentration of lincomycin and tylosin in aqueous solutions. It was observed that low concentration of antibiotics can be determined using microwave sensors and high concentration using optical spectroscopy and capacitive methods. Though optical sensors provide highly sensitive results, the interference due to the presence of other chemicals make the instrumentation complex. In a study of penicillin detection in complex liquid media, Reflectometric Interference Spectroscopy technique was used by passing light to multiple thin layers to study the molecular interaction of assay and surface as the matrix includes milk, surface water etc. The surface modifications are performed by a linker that demonstrated good binding sites [74].
Many fluorescence aptasensors [75] have proved to have potential application in identification of presence of antibiotics such as tetracyclines, chloramphenicol [21], kanamycin [22] etc. along with nanoparticle coating. Most reviews states that electrochemical and optical sensors serve much better option in the fabrication of sensing devices with high sensitivity, miniaturised size and fast response [76].

Mass sensitive biosensors
Most commonly used mass sensitive biosensors involve piezoelectric sensors, in which the crystal is coated by the biosensing material. Piezoelectric sensors generate electricity according to the mechanical force applied on them ( Figure S5). When a biorecognition element is coated on the crystal and placed in contact with a specific analyte, the mass on the crystal increases according to the binding, ultimately reducing the crystal oscillating frequency. A recent work described that high sensitivity and fast response can be obtained by using Quartz crystal microbalance (QCM) in detecting penicillin in food items, as it involves generation of electric signals at the output [77].
Piezoelectric biosensor is cost-effective and is also efficient in producing a real-time output. The design can be improved to obtain good throughput by using a cantilever type, where biorecognition elements are coated. A recent review on the micro-and nanocantilever design explains the various aspects of cantilever design used in biomolecular detection, drug resistant bacteria detection and disease diagnosis [78]. According to the bending caused by the binding of analyte in microcantilever, there will be a deflection in the transduced signal. Along with electrical and mechanical properties, micro cantilever design is also susceptible to thermal properties, due to which an on-chip temperature sensor and temperature compensation need to be incorporated along with the cantilever. An appropriate example is in gentamicin detection from blood samples, which when compared with existing hospital instrument showed a strong correlation among each other [23].

Nanomaterials-based antibiotic biosensors
Nanomaterial-based biosensors are an emerging field in the development of biosensing systems mainly due to the advantages of having larger surface area and their exceptional physical properties. They exhibit good electric, optic, magnetic and chemical properties due to which they are excellent options for biosensing applications. Carbon/Graphene/ Carbon nano tubes (CNTs), quantum dots (QD), metal/metal oxide nano materials are a few nano materials which are widely used in antibiotic detection in recent times [79] ( Figure S6). CNTs are good in detecting small amounts of analyte in the sample. The distinguished electronic, electrochemical, mechanical, thermal properties of graphene and graphene oxide (GO) nanosheets [80] facilitates them to be used as biosensors with good biocompatibility. They are efficient in detection of pathogens, organic compounds and toxic heavy metal ions and hence prove to be a promising option in detection of contaminants in environmental sources [81].
In the detection and quantification of Cloxacillin, an electrochemical nanosensor using graphene oxide -gold nanocomposite showed better stability, sensitivity and repeatability. For the fabrication of highly sensitive sensor, Molecular Impregnated Polymers (MIPs) provided better adsorption of target molecule and were used along with screen printed electrodes [24]. In many of the recent works, nano MIPs are used as the sensing element in biosensors by the development of biomimics. Several studies are based on the molecular imprinting polymers, where nano spheres are immobilised as biomimetic receptor interface in detection of penicillin [82,83]. Magnetic transduction is another best method used in detection of antibiotics, where low magnetic signal is passed in the background. For this reason, magnetic nanoparticles (MNPs) are also used along with electrochemical and optical biosensors for improved sensitivity and selectivity [84]. Electrochemical sensors in which a combination of Printex 6 L carbon nanomaterials and CdTe QDs were coated were used for amoxycillin detection in diverse liquid samples such as milk, synthetic urine and pharmaceutical samples. The experiment resulted in better sensitivity, linearity and proved better analytical performance with less interferences than using any other nanomaterials [25]. Nano materials along with QCM provide better sensitivity and selectivity. One example is by depositing gelatin assisted CuO nanoneedles, onto QCM, precise results with good sensitivity and a low LOD of 9.4 nM were observed in rifampicin detection in urine samples [26]. Electrochemical biosensors with hybrid nanowires have shown good performance characteristics in simultaneous detection of multiple antibiotics in a sample. The electrodes were formed by multisegmented metal nanoparticles on which respective biosensors were immobilised [27]. Aptamer-based electrochemical biosensors using AgNP ink printed electrodes were also tested for use in low-cost disposable antibiotic detection systems [28]. Gold nano particles are found sensitive to β-lactam antibiotics with remarkably low LOD, for making the sensor more sensitive. Sensitivity of the sensor can further improved by using plasmon resonance technique by immobilising AuNPs on their surface [13]. Nanosensors had also been proven to be an optimal option for miniature sensing devices even in environmental water monitoring. One example for the same is in detection of oxytetracycline in environmental water samples, electrochemical sensors are formed by immobilising aptamers in Single walled Carbon nanotubes (SWCNTs). It was observed that this biosensor gave better sensitivity and responsivity as compared to colorimetric, electric and mass sensitive type biosensors [14]. Though research in the field of biosensing methods using nanomaterials has proved to be very effective in the production of portable microsystem, a challenge in development of antibiotic detection kit for on-site applications persists.

Fabrication of biosensing system using lab-on-chip technologies
The emergence of lab-on-chip (LOC) technology wherein one or more laboratory process can be integrated on a single chip, has brought a tremendous change in the field of miniaturisation of equipment/devices [85]. The application of this technology is advantageous in employing physical principles such as laminar flow of fluid, thermal transport, diffusion etc. involved in several biochemical reactions. This enabled LOC technologies to prove their excellency in the field of diagnostics, genomics, proteomics, biosensors and cell research [86]. Most of these operations can be incorporated with the electronic control (as shown in Figure 2) and hence it has a wide application in the biomedical field as it helps in diagnosis, chemical analysis and synthesis, environmental monitoring etc. to be performed on a miniaturised scale.
The liquid management tasks in laboratory include initiating reaction, mixing, filtration, dispensing, measurement etc. Due to the low cost of polymer materials, glass and silicon are generally used in fabrication of lab-on-chip platforms [87]. With a less sample volume, these processes become more difficult, therefore Micro Total Analysis Systems (μTAS) plays a great role in assisting such processes in laboratory. This technology aids in the analysis of various functionalities in applications ranging from fluid control, fluid separations, detection of various analytes in sample, drug screening, disease diagnosis, protein analysis etc [88]. Microfluidics is a subset of μTAS, along with the integration of small fluid flow channels, microscale valves and pumps that deal with the study of a very small amount of fluid flow through microchannels, their behaviour and precise control [89]. As this technology involves less sample consumption, the reagent cost and production of waste can be reduced and due to the smaller size, fast response is obtained with better control of process [90].
To ensure precision engineering, lithographic techniques such as soft lithography, photo lithography and nano-imprinting technologies [91] are used for the fabrication in micro or nanoscale, as it handles fluid in picolitres/nanolitres/microlitres scale. This technology is advantageous in improving the efficiency of point-of-care (POC) devices. A challenge while using this technology is the complexity in microfluidic system design automation [92]. In microfluidics, the fluid flow depends on the viscous force than any other convective forces or gravitational forces [93]. The fluid flow in such systems can also be initiated either by capillary force or by the integration of valves or pumps on the chip [94]. The electronic control for pumping and valve mechanism can be provided by Micro-Electro-Mechanical System (MEMS) technologies. The combination of μTAS with MEMS had been facilitated in simultaneous monitoring of different parameters using a single system. One such study has been performed in identification of several microorganisms, antibiotic residues and neutrophils in raw milk by antibody microarray [95]. Such technologies serve as precursors to develop more economical, time saving and simpler analytical methods in food and environmental monitoring systems. The detailed review of recently used fabrication materials and techniques was published by Bruce and team that mentions the moulding, 3D printing and nano-fabrication techniques [96].

PDMS-based microfluidic systems
Microfluidics can be fabricated using silicon/glass hydrogels, paper or thermoplastics [97]. Mostly, polydimethylsiloxane (PDMS) is the material used in fabrication of microfluidics because it is chemically inert, biocompatible, cheap, transparent, and leak proof. The microchannels for fluid flow are usually imprinted in the PDMS and can be sealed either using PDMS layer or any other inert polymeric substances. Plasma bonding is used for sealing of microchannels with glass. The microchannels are formed by using replica moulding, in which a patterned reusable PDMS mould is used along with the polymer. Another moulding technique in PDMS fabrication is capillary moulding where the PDMS mould is made contacted along with the substrate and a liquid polymer is filled in patterns. After curing, the mould is removed slowly and forming the solid microstructures [98]. The main steps involved in PDMS fabrication are depicted in Figure 3.
The hydrophobic nature of PDMS material resist the flow of fluid through microchannels, which leads to the requirement of incorporating micro valves and micropumps in the system and thus increases the complexity of the system. As mentioned above, many recent studies incorporate nano electromechanical systems (NEMS) and micro electromechanical systems (MEMS) along with the microfluidic structure for ensuring the proper flow of fluid through microchannels. Mostly, the pressuredriven or electro-kinetic methods allow the fluid to flow through the channels. The former method requires an additional vacuum source or pneumatic pump, whereas the latter works based on the movement of molecules based on the electric charges. Many valve mechanisms can also be provided for efficient fluid flow through microchannels using these two principles [99]. Through microchannels, the fluids can be pumped using pneumatic system with a pressure of nearly 350 kPa. The transduction methods include incorporation of biosensor along with the microfluidics. Optical biosensors are most commonly used in sensing applications. One example of detection of aminoglycoside using chemiluminescence is due to the complex formation with Cu(II) and antibiotic [100]. For pumping of sample and reagents to the microfluidic structure, syringe pumps are used that have an advantage of delivering accurate volume of fluid at precise rate, ranging from 0.8-5.0%. A pneumatic actuated pumping mechanism is used in detection of presence of antibiotics in sea water [101]. Another efficient approach is by using a vacuum pump with constant flow rate [102] for pumping fluids through microchannels in nL, which is not bulky and can be attached to the microfluidic structure, irrespective of the application. Here a syringe is used and this method is well suited for hand held PDMS devices. Although this technology is cost-effective and compatible with almost all assays, a few challenges still exist, such as incorporation of pumping mechanism due to the hydrophobic nature of the material, minimising clogging of microchannels due to precipitation or small particles etc.

Paper-based microfluidics
Paper-based biosensing technologies are widely used now-a-days in low cost applications. They provide cheap, portable and disposable techniques suitable in analysing variety of contaminants in water and other liquid samples [103]. Development of microfluidic paper-based analytic devices (μPADs) is an innovative area of interest for most researchers. For paper-based devices, commonly used types of paper are nitrocellulose membrane, cellulose chromatography paper, cellulose filter paper, printing paper (Whatmann filter paper, Whatmann chromatography paper of different grades, Millipore, etc). These papers are differentiated by their flow rate and thickness. Different techniques like wax printing, screen printing, cutting by punching were used in the fabrication of paper-based devices. Table S7 describes the advantages and disadvantages of different fabrication techniques in μPADs [104][105][106][107][108][109][110][111][112][113][114][115]. The paper was printed on both sides using wax and then heated at a particular temperature for a specified time, in wax printing, this allows the paper to form hydrophobic regions and the hydrophilic regions to act as microchannels for fluid flow. Screen printing is mostly used in integrating electrodes with paper for electrochemical sensing [116]. Though colorimetric detection is most commonly used in μPADs, selectivity issue persists in using colorimetric reagents and hence masking of interfering metals becomes mandatory [117].
Miniaturisation of μPADs was made possible by using smaller volumes of analytes and sample with increased rigidity. The standard μPADs were immersed in varying concentrations of sodium periodate (NaIO 4 ) at different time intervals and the optimal miniaturisation parameters were studied [118]. Paper microfluidics was also used as a technique for detecting different classes of antibiotic, with a very less amount of sample volume. Using coordination chemistry principle, the antibiotics are capable of binding with metal ions which can be identified as a colour change in a filter paper. This colorimetric sensing technology was used to detect the oxytetracycline (with Cu 2+ ) and norfloxacin (with Fe 3+ ) concentration in spiked pork samples [119]. Recent studies are focused on more reliable technologies for detecting antibiotics using smart phone. Image processing techniques can be applied to the color obtained from the sensor by integrating with smart phone technology. Li and team performed an experiment for the detection of tetracyclines using paper based ELISA testing [120]. An android app was developed to detect the colour change, which gave the information regarding sensing data. This technology enables the device to be used in on site applications too. One of the major limitations of using paperbased technology is to improve the limit of detection and sensitivity. Use of 3D slip-PADs had found to be an efficient option for the improvement of sensitivity by providing multiple fluidic paths for the easy delivery of different fluids [121]. This method enables enhanced release and mixing of reagents and also eases usage of the devices even by an unskilled operator too.

Silicon-on-chip technology
This technology involves fabricating the chips with integrated silicon sensors, due to which contamination in the microchannels can be avoided to an extent. Photolithography is involved that ensures mass manufacturing of the device with low cost [122]. The photonic crystal sensor chip is fabricated on a silicon wafer. A microfluidic approach involving photonic crystal micro cavities along this technique was used to detect gentamicin, an aminoglycoside class of antibiotic with high specificity and good detection limits from 0.1 nM to 1 μM [123]. Though silicon-based lab-on-chip technology is advantageous in low cost production of portable biosensing devices, its sensitivity along with miniature integration technology remains a challenge [124].

Simulation-based studies for the development of microfluidics
Software simulation-based studies play major role in designing and developing miniaturised monitoring devices. Computer Aided Design (CAD) is commonly used for designing the models for fabrication. The lab-on-chip devices are designed using AutoCAD, which is very useful in formation of numerous engineering geometries. Replica moulding and masking are done using AutoCAD software. These methods are used in photolithography to photoresist the the wafer surface and in soft lithography to generate the moulds by which the desired pattern can be repeatedly fabricated. The mask design is one of the crucial step in achieving accurate and precise result [125]. This section highlights some of the important software modelling tools employed for efficient biosensor design.
An Extensible Simulation Package for Research on Soft Matter Systems (ESPResSo) is a python-based open source software to analyse the molecular dynamics of the systems involving biological, physical and chemical research [126]. It helps in solving the hydrodynamic interactions, bonded and non-bonded potentials and also involves fast methods for electro and magnetostatics with the help of advanced algorithms. This software was used in studying the fluid motion through microchannels modelled by lattice-Boltzmann method to study the cells present in blood flow based on the principle that each blood cell has different mass and elasticity. For example, the RBCs were modelled by tuning many parameters based on their properties, and their collision with other cells was studied and analysed by considering the blood flow through microfluidics and the effect of external force on volumetric flow rate [121]. Kovalčíková and team employed three different topologies from simulation box and found that there exists a linear relationship between the applied external force and volumetric flow rate of cells present in blood [127].
Computational Fluid Dynamics (CFD) simulations help to understand the behaviour of flow through patterns/structures, which are practically difficult to observe and analyse. One of the softwares that aids in analysing fluid dynamics through microchannels is the Flow3D software. For the numerical modelling of microfluidic cells, the interaction studies of the fluid with surface and fluid transport mechanism through microchannels are also carried out [128]. Flow3D is a good platform to study the physical properties of numerous reagents involved in analytical problems. The motion of these reagents can be simulated, the physical changes occurring during their mixing and interaction can also be studied using this software [129].
COMSOL Multiphysics software platform can be used to study the physics of a system related to fluid flow mechanics, thermodynamics, chemical reaction studies etc. Using this software, microfluidic systems can be designed and simulated prior to fabrication, so that its design can be optimised. This software works on the basis of Finite Element Method (FEM), using which numerical methods are employed for solving engineering problems including structural analysis, mass transfer fluid flow, heat transfer, reaction chemistry, parameter optimisation etc [130]. In a study on the analysis of pharmaceutical residues present in waste water, a microfluidic structure with electrochemical biosensor was employed [93]. The velocity distribution in microchannels was studied by solving Navier-Stokes equations. Figure S8 shows the simulation window of COMSOL Multiphysics software microfluidic applications.

Conclusion and future scope
AMR is one of the global threat due to the wide spread use of antibiotics, it becomes mandatory to follow regulations on extravagant use. Even though many successful research works have been carried out employing recent techniques for antibiotic monitoring, no commercial sensing devices has yet reached the market. Future efforts need to focus on portable, miniaturised, generic devices that can be used in industries where antibiotics are widely used and expelled to the environment without infringing any regulatory measures. By making use of in-situ monitors and setting a threshold value for the antibiotic release to the environment, effective measures for the reduction in antibiotics present in the environment can be employed and there by the AMR issue can be reduced to a great extent.