A Review of Fabrication Techniques and Optimization Strategies for Microbial Biosensors

Challenges of stability and specificity associated with early generation sensors necessitate the fabrication and optimization of microbial biosensors. More so, the global biosensors market size currently valued at USD25.5 billion in 2021 is expected to grow at a compound annual growth rate (CAGR) of 7.5% to USD36.7 billion in 2026. Microbial biosensors are bioanalytical systems that integrate microorganisms with a physical transducer to generate signals, thus, aiding the identification of analytes. The biosensors are fabricated through a series of steps comprising microbe selection, immobilization onto a matrix, microfabrication, calibration, and validation. The transducers integrated microorganisms generate quantifiable signals, enabling real-time monitoring of a diversity of analytes within food samples. The optimization strategies are scrutinized, with a particular focus on the integration of sundry nanoparticles, such as magnetic, gold, and quantum-dot nanoparticles, which enhance sensor performance. Distinct advantages offered by microbial biosensors promise to revolutionize food quality assessment via cost-effectiveness, rapid sample testing, and the ability to provide access to real-time data. Literature have highlighted certain limitations including interference from complex matrices, instability of microorganisms, and microbial lifespan. In assessing their economic importance, a comparative analysis is presented against conventional food analytical methods like ELISA, PCR, and HPLC; thus, highlighting the unique strengths of microbial biosensors. The future perspectives focus on the potential of the technology in addressing the need for continuous monitoring challenges, and research for further improvements in the biocompatibility of fabrication processes and long-term reusability.


Introduction
Biosensors are devices that quantify biological or chemical reactions by generating signals proportional to the concentration of an analyte in a reaction [1].The foremost biosensors were created by Leland Charles Clark Jr. in 1956 and it was intended to assess the oxygen content in blood [2].Following that, researchers from several disciplines have collaborated to create better advanced, reliable, biosensing devices for use in every human field [1].Biosensors identify and transform biological molecules DNA and RNA, antigen, protein, complete cell spoilage precursors and microbial contaminated by-product) into optical, electrochemical, piezoelectric, nanomechanical and mass-sensitive signals [3].Biosensors operate on the principle of signal transduction and element biorecognition by measuring reactions biologically or chemically and producing signals commensurate to the concentration of an analyte in the reaction [4].
Biosensors contain biological component that helps the device recognize or communicate with the analyte [1].This recognition generates a biological, chemical or physical response which can be can be optical, thermal, electrochemical, acoustic, or magnetic, which is then converted into an electrical signal by a transducer [4].These signals, collected by the transducer are then transmitted to an electrical device (amplifier, processor) for conversion into a readable format after which the output is displayed [5].
Microbial biosensors are bio-analytical devices that uses living microbial cells to detect and measure the presence of specific substances called analytes in various environments [6].Their purpose is to harness the potential of microbes to react to certain stimuli or generate quantifiable reactions when particular chemicals or conditions are present Their purpose is to harness the potential of microbes to react to particular stimuli or generate quantifiable reactions when particular chemicals or conditions are present.Many methods, including modifications to the expression of a gene or genes, the synthesis of fluorescent proteins, or the emission of specific metabolic byproducts, can be used to achieve this.Although early microbial biosensors were less expensive than enzyme-based biosensors, they lacked the great degree of selectivity of the latter.
But when genetic engineering became a reality, everything changed, and microbial biosensors quickly advanced [7].Many methods, including modifications to the expression of a gene or genes, the synthesis of fluorescent proteins, or the emission of specific metabolic byproducts, can be used to achieve this.Although early microbial biosensors were less expensive than enzymebased biosensors, they lacked the great degree of selectivity of the latter.But when genetic IOP Publishing doi:10.1088/1755-1315/1342/1/0120153 engineering became a reality, everything changed, and microbial biosensors quickly advanced [8].By making use of new molecular techniques, microorganisms can now be genetically engineered to contain foreign proteins, receptors or enzymes that produce a recognizable and quantifiable signal when exposed to a particular target analyte [9].These interact with the analyte and thus, triggers a biological response that generates a measurable signal that can be quantified and measured [11] [12], as depicted in Figure 1.In healthcare, microbial biosensors can be used in the rapid detection of diseases, monitoring of drug efficacy, or in the development of diagnostic tests.The reports of Akyilmaz and colleagues highlighted the fabrication of a microbial biosensor for the determination of epinephrine.This was achieved by immobilizing white rot fungi (Phanerochaete chrysosporium ME446) in gelatin, with glutaraldehyde as the crosslinking agent on a Platinum electrode [16].A graphenebased biosensor used for the early detection of iron deficiency in the saliva has been developed by Oshin and colleagues [17].This biosensor used anti-ferritin antibodies and a linker molecule (1-pyrenebutanoic acid, succinimidyl ester) to functionalise the graphene-based field-effect transistors (GFETs).This facilitated specific conjugation with ferritin antigen [17].Additionally, they can be essential tools in industrial processes, such as fermentation, where they help optimize conditions or detect the presence of contaminants and help in continuous monitoring of the food product.The reports of Ahuekwe and colleagues highlighted the use of biosensors based on nano-chitosan materials for barcoding, primarily as colorimetric sensors that indicate changes in temperature, freshness, or pH of packaged foods [18].In the same vein, an ion selective  [19].More so, genetically modified Saccharomyces cerevisiae strain have been incorporated in the quality assurance of milk.The yeast was used as the bio-element of the microbial biosensor for the detection of zearalenone in milk [20].

Fabrication Steps of Microbial Biosensors
Fabrication techniques for biosensors vary depending on type, the target analyte, and the applications [21].As shown in Figure 3, the fabrication of microbial biosensors requires several crucial processes which includes:

Selection of Microorganism:
The selection of microorganisms in the fabrication process of microbial biosensors is crucial as it directly impacts the functionality and effectiveness of the biosensor.Among other factors There are some important factors to consider and steps to take when choosing the right microorganism for microbial biosensors.It is very necessary that the analyte to be detected should be specified (e.g., heavy metals, organic compounds, mycotoxins, antibiotics, etc.) [22].Microbial biosensors use the interaction between the analyte and the microorganism to identify analytes.As a result, the microbe of choice should be selected based on its capacity to identify the desired target analyte with precision [6].Various microorganisms possess natural receptors, enzymes, or proteins or receptors that can be genetically modified to interact with specific analytes, allowing for accurate and selective detection [7].
Since every microorganism has a unique metabolic pathway, they display varying degrees of sensitivity to an analyte.The best microorganism to use when fabricating a biosensor is one that exhibits high sensitivity and can produce a detectable response even at low analyte concentrations.[2].It's important to take into account the biosensor's response time.Fastresponding microorganisms can provide more precise real-time qualitative and quantitative data regarding the presence of an analyte [23].Variations in the analyte concentration range, operating settings, and environmental factors can all affect a microbial biosensor's stability, efficacy, and functional lifetime.Selecting a microbe that can withstand these circumstances while still being able to recognize and react to the analyte is crucial.Lastly, microbes can be genetically altered to change the properties of their response or to improve their affinity for the analyte or sensitivity [2].Making the genetically modified microbe choice for the specific IOP Publishing doi:10.1088/1755-1315/1342/1/0120156 biosensor it will be used for makes the customization and optimization of the biosensor for its particular requirement [24].

Immobilization:
Immobilization is fundamental to the stability, usability and long-term efficacy of microbial biosensors.It cannot be neglected from consideration at any time while designing them [25].. Immobilization refers to the adhesion of microorganisms onto a surface or into a matrix so that they can be used with stable and reusable biosensors [26].This is because the microorganisms are immobilised with a suitable support material attached to complementary electrode, gel or membrane.The type of immobilization used may differ depending on what kind of organism is involved and different surface materials suited for this purpose can be found.The four immobilization methods are: adsorption, covalent bonding, cross-linking and encapsulation [27] (Figure 4).Adsorption stands out as a straightforward technique, as it circumvents the need for extensive pretreatment of sensor compounds or the use of specialized chemicals.Various materials such as activated-carbon, alumina silica gel, collodion, cellulose, hydroxylapatite, glass, clay, and innovative substances can adsorb enzymes without altering their inherent configuration.In adsorption, the biological material adheres to the surface through ionic interactions, van der Waals forces, Coulombic forces, or hydrogen bonding [28].In the immobilization of cellular structures, the strong adsorptive adhesion of cells to a previously treated polymer surface ensures that removing the surface cells results in their disintegration.The efficacy of adsorptive immobilization is predominantly influenced by the surface properties of the transducer, including the presence of polar groups, its charge, energy homogeneity, and redox potential [8].However, the adsorption method has limitations when it comes to accommodating high concentrations of biomaterials.To augment the adsorption of biomaterials, transducers are often pretreated to introduce charged or polar groups, enhancing the adsorption of bio components [28].
The enhancement is achieved through various oxidation processes or surface modifications using functionalizing chemicals or polymers.For instance, the oxidation of carbon electrodes and gold increases adsorption capabilities for nucleic acids, proteins, and microbes [29].Chemisorption, in particular, results in much stronger bond between the support and the bio-component.Adsorption formed identification elements exhibit high sensitivity to changes in temperature, pH, substrate concentration, and ionic strength [31].This technique is typically employed in situations where the weak interaction between the transducer and the bio-component suffices, and the sensor is utilized for brief periods [27].

ii. Cross-linking
The cross-linking immobilization technique entails connecting biological material to a gel or solid substrate using a bi-functional compound like glutaraldehyde.This chemical operates by forming Schiff bases with the hydroxyl, amino, and thiol groups found in proteins or nucleic acids.Numerous polymers, including cyclodextrins, gelatin polyvinyl chloride agar, polyacrylamide, and various other polymers and gels, have been explored as matrices for bio-materials.However, none proved to be ideal, as the encapsulation IOP Publishing doi:10.1088/1755-1315/1342/1/0120158 process could lead to slow diffusion of the substrate through the resulting material.
Additionally, this method has the drawback of yielding compounds with suboptimal mechanical properties.Nevertheless, it could be employed to enhance the stability of adsorbed bio-materials [30].
iii.Covalent bonding This is widely employed as an immobilization technique, involving the establishment of a covalent bond between a support and a bio-component.The selection of chemicals is guided by the characteristics of the support material and the target molecules for binding [28].The process of covalent bonding typically unfolds in three stages: the initial step focuses on purifying or clarifying the support and introducing relevant functional groups to its surface; the second, involves the binding of biomaterial; and the final step entails the removal of weakly attached molecules through solvent extraction [31].Sensors constructed from carbon, metals (such as silver, gold, or platinum), polysaccharides (including cellulose and its derivatives), glass, poly(methyl methacrylate), nylon, and compounds containing imidazole groups or free SH, -NH2, or -COOH groups are commonly used [28].Covalently attaching proteins with nucleophilic functional groups in their side amino acid chains does not compromise enzyme activity.Covalent bonds are most effectively formed under optimal temperatures, pH levels, and ionic strengths, with the specific substrate used shielding the active site of an enzyme-based process.When oligonucleotides and DNA are crosslinked to chitosan, a series of amide connections is established, facilitating the immobilization of oligonucleotides and DNA [28].Terminal nucleotide residue modification is a recurring phenomenon.Thiol groups incorporated into nucleotide residues enable the formation of uniform layers of oligonucleotides, typically oriented orthogonally to the surface, through chemisorption on gold.The primary advantage of this covalent binding is its ability to establish a robust connection between the biocomponent and support, preventing biocomponent loss [27].

iv.
Entrapment: Entrapment involves confining biological materials within a designated space [28].This confinement permits the passage of substrates and products while retaining the biomaterial.Consequently, post entrapment, the biomaterial remains unattached to the supporting structure, with the support serving as a physical barrier to prevent its diffusion [32].This, opens up new avenues in material science, particularly with the utilization of inorganic/organic hybrid polymer matrices for immobilizing enzymes [28].This method capitalizes on the support's attributes, such as uniformity, high purity, biocompatibility, thermal chemical stability, while also offering the advantage of gentle processing conditions that minimize the risk of damage to the biomaterials [33].Notably, entrapping biomolecules within a sol-gelmatrix has proven to be an effective technique for immobilizing a wide range of biomolecules [34].

Microfabrication:
Microfabrication techniques play a significant role in-the fabrication of microbial biosensors, enabling precise control over sensor design, miniaturization, and integration with other components [12].These techniques can be used to create microscale channels and chambers for the microorganism to reside in.These microscale structures can be integrated with a transducer to produce a measurable signal [35].Some commonly used microfabrication techniques in the fabricating microbial biosensors are: i. Photolithography: Photolithography is a well-established technique used in microfabrication processes [36].It involves the use of materials known as photoresists to create patterns on a substrate.In the fabrication of biosensors, photolithography can be employed to generate microscale features and structures such as channels for fluid flow or electrodes for sensing on the biosensor platform [37].This technique offers control over feature size and placement ensuring patterning of electrodes, channels, and other components on biosensor substrates as the ability to achieve resolution is crucial in designing sensors with details [38].Furthermore, photolithography has a high degree of scalability, which makes it appropriate for large-scale manufacturing when required in specific applications [39].Because photolithography techniques are complex, it takes skilled operators to complete the necessary steps in an efficient manner.Additionally, the equipment used in photolithography can be costly [38].
ii. Soft lithography: Replica molding and microcontact printing are two techniques commonly used in lithography to create microscale structures and patterns [40].These methods involve transferring patterns from a master mold onto a biosensor substrate using materials such, as polydimethylsiloxane (PDMS) [41].Soft lithography is often employed to fabricate channels in biosensors allowing controlled flow of analytes and microbial cells.Its ability to produce high resolution patterns enables the creation of IOP Publishing doi:10.1088/1755-1315/1342/1/01201510 microstructures and precise biosensor components, which are crucial for achieving optimal functionality [42].PDMS, being biocompatible is particularly well suited for biosensing applications involving samples and cells [41].However, it's important to note that soft lithography may not be suitable for all applications that require substrates or specific material properties since it primarily utilizes elastomeric materials like PDMS [43].Additionally the complexity involved in aligning and bonding components during the fabrication of multi-layered devices using soft lithography can pose challenges, in certain cases because of its complexity [44]. iii.
Thin-film deposition: Thin-film deposition techniques, such as sputtering or chemical vapor deposition, are used to coat the biosensor substrate with various functional materials [45].For instance, electrodes for electrochemical microbial biosensors are frequently deposited using thin metal films, such as platinum or gold.They enable the identification and quantification of electrical signals generated by microbial metabolic processes They enable the identification and quantification of electrical signals generated by microbial metabolic processes [46].With thin-film deposition techniques, the thickness of deposited films can be closely controlled.With thin-film deposition techniques, the thickness of deposited films can be closely controlled.[47].This is important because the thickness of the film can affect the sensitivity and response of the biosensor in its design [47].These methods usually yield very uniform films, guaranteeing reliable sensor performance over a wide surface area [48].The process of thin-film deposition can be complex and time-consuming, involving multiple steps to ensure precise conditions [46].Maintaining quality control and reproducibility can also be difficult.Certain materials used in thin-film deposition might not work well in biological systems or might need further functionalization processes in order to be considered biocompatible [49].
By manipulating the arrangement and positioning of these cells using printers we can ensure sensing efficiency.Furthermore, bioprinting enables the creation of structures that mimic real microbial habitats thereby enhancing the performance of biosensors.With bioprinting we can accurately position biomaterials and cells to develop biosensors tailored for applications [51].This accuracy comes in especially handy when building  [52].Miniaturized biosensors with high spatial resolution can be created through bioprinting, which makes them appropriate for uses where size is a crucial factor, like wearable or implantable devices [53].It can be challenging to maintain cell viability both during and after the bioprinting process, despite the benefits.Cell health may be adversely affected by the mechanical and environmental stresses associated with bioprinting [54].Additionally, using bio-printing techniques are not costeffective [55].
Choosing the right microfabrication technique depends on factors related to the biosensor, such, as the type of microorganism, the specific substance being analysed, and how it will be used.
With the help of these techniques, scientists can create better biosensors that are more sensitive, selective, and compact, which makes them useful for a range of applications.

Calibration and validation:
Establishing the connection between analyte concentration and observed signal is done by calibrating biosensors with known concentrations of target analytes.Later, experimental samples are used to test the biosensor and its results compared with reference methods in order to validate it [56].
Among the many factors to consider in making microbial biosensors, an especially important one is selecting a suitable specimen of cells as receptors.These construction processes can be used singly, or in combination to manufacture microbial biosensors for different purposes [35].

Optimization of Microbial Biosensors
Microbial biosensors have a range of applications.However, to ensure their effectiveness, in these applications it is crucial to optimize them for stability, sensitivity and specificity.An important aspect of optimizing biosensors for use in the food industry is the selection of the appropriate microbial strain for detecting specific analytes.For example, Saccharomyces cerevisiae has been utilized in developing biosensors to detect ethanol in beverages while Lactic acid bacteria has been employed in creating biosensors for identifying lactose, in dairy products [8].

The Integration of Microfluidics for the Optimisation of Microbial Biosensors
The use of devices enables the miniaturization of reagents and samples which's particularly beneficial, for portable or point of care (POC) applications.This leads to a decrease in the dilution effect and an enhancement, in the concentration of target analytes thereby improving the sensitivity of biosensors [57].Furthermore, the flow of the analyte can be carefully controlled through channels ensuring a regulated delivery to the biosensor.This approach minimizes the requirement for reagents and reduces waste generation.[12].These systems have a range of uses as they can be adjusted in size depending on the requirements.Scientists have created biosensors that can detect types of harmful bacteria found in food such as E. Coli O157;H7 and Salmonella when testing food samples.[6][60].The use of microfluidics makes it easier to perform continuous operation and monitoring in real time.Multiplexing of biosensors or sensor arrays can be done using microfluidic devices that integrate these sensors onto a chip [59].This increases throughput and efficiency by allowing for the simultaneous analysis of many different samples or analytes.Furthermore, continuous flow configurations can be made in microfluidic channels, allowing for the continuous delivery of analytes to the biosensors [60].Integration with microfluidics also makes on-chip cell encapsulation and immobilization possible [61] (Figure 5).Microfluidic devices can encapsulate or immobilize microbes (i.e protect them from shear stress and provide a zone of stable microconditions).This increases the shelf-life of microbial biosensors and stabilizes their operational effectiveness, enhancing viability [62].

The Integration of Nanotechnology for the Optimisation of Microbial Biosensors
The creation of meticulously arranged particles made of atoms that have the ability to alter their material properties at a specific size between 1 and 100 nm is known as nanotechnology [63].
The application of nanotechnology to the production of biosensors is currently a method used by researchers in an effort to make microbial biosensors more stable, sensitive and specific.Due to their nanoscale sizes, various nanomaterials-including carbon nanotubes, nanosheets and nanofibers-have shown unique chemical and physical properties.For instance, their exceptional optical, electrical, and magnetic properties coupled with high interfacial reactivity and huge surface to volume ratios can increase the biosensors 'stability, sensitivity, and selectivity.The novel optical electrical, magnetic and mechanical characteristics of these biosensors can be exploited to create highly selective and sensitive microbial detection devices [66][67].Their optical, electrical, magnetic and mechanical properties can be exploited to develop highly selective and sensitive microbial detection devices [6] iii.Magnetic nanoparticle-based biosensors: Magnetic nanoparticles (MNPs) have remarkable magnetic properties and are commonly used in the design of microbial biosensors [69].MNPs can, therefore, serve as a recognition element to enhance the sensitivity and specificity of microbial detection.They can also be used to increase the selectivity of the biosensor and select out just target analytes from complex samples. [70].
iv.Quantum dot based biosensors (QDs): QDs can be used to detect food additives, pathogens heavy metals, nutrients antibiotics, and insecticide residues [71].Due to their antioxidant, antimicrobial, and inhibitory qualities, they can also be utilized in packaging materials to extend the shelf life of products, inhibit the growth of microorganisms, enhance mechanical qualities, block gases and UV light [72].
Additionally, microbial biosensors' stability can be increased by nanoparticles.For instance, a microbial cell can be shielded from adverse environmental factors like high pH by incorporating silica nanoparticles into it.In a similar vein, the biosensor's shelf life can be extended and degradation prevented by using polymer-coated nanoparticles [67][75].

Comparison with Other Analytical Methods
Microbial biosensors offer distinct advantages and disadvantages when compared to other conventional methods of analytical processing such as ELISA, PCR, and HPLC, such as enzyme-based biosensors or chemical assays.In Table 1, a comparative analysis of some of their properties is presented.
Not cost-effective [85] On-site testing Possible for on-site and in-field testing [89].
Cannot be employed for onsite or emergency applications [90].
Cannot be employed for onsite or emergency applications [91].
Cannot be employed for on-site or emergency applications [92].

Quantitative Analysis
Can be used for quantitative analysis and qualitative analysis.
Can be used for quantitative analysis and qualitative analysis [83].
Can be used for quantitative analysis and qualitative analysis [93].
Can be used for quantitative analysis and qualitative analysis [94].

Figure 5 :
Figure 5: Miniaturized biosensors based on existing handheld devices and microfluidic systems for point-of-care testing (POCT) [12].

Table 1 :
Comparative Analysis Between the Properties of Microbial Biosensors, ELISA, PCR and HPLC