Perspective—Surface-display Techniques in Electrochemical Biosensor Designs for Health Monitoring

Enzymatic and microbial electrochemical biosensors integrate enzymes and microorganisms as biological recognition elements into the sensor design and functionality. Enzyme-based sensors offer high sensitivity and selectivity for target analyte detection. However, these have limited stability necessary for continuous analyte monitoring. Contrarily, microbe-based electrochemical sensors provide a means for continuous analyte sensing but are associated with challenges related to analyte selectivity in complex samples. To address these limitations, surface-display methods, which bind enzymes to microbial surfaces, enhance biosensor selectivity and sensitivity. This perspective outlines the application of surface-display techniques, offering a promising avenue for health monitoring.

Electrochemical biosensors (EBs) are analytical platforms that integrate a biological recognition element with an electrochemical transducer, or electrode, to produce a measurable signal proportional to the analyte concentration.EBs have demonstrated vast potential as devices in medical diagnostics and health monitoring.These devices have been designed for the detection of clinically relevant species, such as uric acid, lactate, cholesterol, dopamine, creatine, and nitric oxide. 1 Furthermore, glucose monitoring devices for diabetes patients are an effective, commercially available EB capable of continuous, in vivo measurements. 2In addition, EBs are powerful tools for detecting infections at early stages, allowing for timely diagnostics and adequate treatment strategies. 3he major components of the biosensor determine its performance, which is characterized by the analytical figures of merit, including limit of detection (LOD), signal-to-noise ratio, selectivity, specificity, linear dynamic range (LDR), and response time.These components generally include (1) a selective biological recognition element, (2) an electrode transducer, (3) a signal processor, and (4) a data display program. 4EBs offer a means for collecting quantitative analytical information by utilizing enzymes, antibodies, aptamers, protein receptors, organelles, or microorganisms as the biorecognition elements to selectively interact with the analyte of interest.Considering the inherent complexity of biological samples, the bioreceptor plays a critical role in analyte specificity and overall analytical performance of the biosensor.The bio-recognition element must be in direct spatial contact with the electrode transducer, which effectively converts the biological response resulting from the bioreceptor-analyte interaction into an electrical signal relative to the analyte concentration.
EBs can be characterized by the type of biorecognition element implemented in the device design.Namely, enzymatic and microbial EBs employ purified enzymes and whole microbial cells, respectively, as bioreceptor elements.Non-enzymatic and non-microbial recognition elements include other selective biological materials, such as aptamers or antibodies. 1,5In terms of enzyme and microbebased EB designs, each recognition element is associated with distinct advantages, as well as challenges in device performance.Enzyme-based EBs offer high selectivity and sensitivity for target analytes.However, enzymes are prone to denaturing over time, therefore limiting long-term biosensor stability required for continuous analyte monitoring.In contrast, microbial-based EBs provide long-term stability but have challenges associated with selectivity toward a specific analyte.Recent advances in surface-display methods aim to merge the advantages of both enzymatic and microbial-based EBs for improved biosensor designs.These techniques involve binding enzymes to the surfaces of microorganisms, which presents advantages over traditional enzyme immobilization methods.Enzyme surface-display methods offer improvements in EB analytical performance, namely with regard to sensor selectivity (due to enzyme specificity for target analyte), sensitivity, and longterm stability (due to adaptability of microbes to environmental conditions), enabling continuous analyte monitoring in complex biological matrices in the presence of structurally similar species. 6his perspective offers a critical overview of recent advances in both enzymatic and microbial EBs, highlighting the benefits and drawbacks of each biological recognition element for human health monitoring.Additionally, we discuss the future outlook on the utilization of surface-display techniques for health-related biosensing applications.

Current Status
Enzymatic electrochemical biosensors for health monitoring.-Enzymaticelectrochemical biosensors (EEBs) employ enzymes as the recognition element for analyte detection. 7,8Early EEBs utilized glucose oxidase (GOx), coupled with a redox-active mediator to initiate mediated electron transfer reactions for selective, electrochemical detection of glucose.Since this fundamental development of the glucose biosensor, three generations of sensor designs have been explored based on electron transfer mechanisms at the enzymeelectrode interface. 9The first generation involves the reaction of glucose with natural oxygen, catalyzed by GOx, to produce hydrogen peroxide (H 2 O 2 ).The electron transfer reaction of redoxactive H 2 O 2 then produces an electrical current signal proportional to glucose concentration.However, the first-generation EEBs are associated with several challenges that limit biosensor response capabilities, including interferences from other electroactive species within the anodic potential window of H 2 O 2 oxidation and the oxygen dependence of the glucose reaction. 2In the second-generation EEBs, artificial mediators are implemented, shuttling electrons from flavin groups to the electrode surface via mediated electron transfer (Fig. 1A).This process involves a three-step reaction mechanism: (1) glucose is converted to gluconic acid via reaction with GOx, (2) GOx is reduced by the electron mediator, and (3) the mediator is oxidized at the electrode, producing an electrical current output. 10Mediators must display favorable electrochemical properties, pH independence, low solubility, biocompatibility, and stability. 10Common electron mediators employed in GOx z E-mail: osimoska@mailbox.sc.edu = Equal Contribution.**Electrochemical Society Member ***Electrochemical Society Student Member biosensors include ferricyanide, phenothiazines, quinones, and transition metal complexes. 11The use of electron shuttling in the second-generation biosensor design enhances electron transfer and electrochemical signal.However, artificial mediators have challenges associated with electrode surface affinity, limiting biosensor lifetime and stability. 11The third-generation EEBs are mediatorfree, utilizing modified electrode surfaces to facilitate direct electron transfer (Fig. 1A).Successful third-generation biosensor designs yield significantly improved analytical performance, with enhanced electron transfer rates resulting in greater signal-to-noise ratios, high stability, and reduced interferences. 93][14] The electrochemical signal-to-analyte relationship of EEBs also depends on the sensor design methodology.Most EEB devices generate catalytic current from higher electron transfer rates associated with enzymatic redox reaction which directly correlates to analyte concentration.
Nonetheless, inhibitory current measurement has also been implemented in recent EEBs. 15,16Inhibition involves covalent analyte binding to the enzyme active site, which inactivates the enzyme and decreases electron transfer.The resulting lower rates of electron transfer yield less electrical current signal, which is inversely proportional to analyte quantity.Overall, in both catalytic and inhibitory current signals, the analyte is selectively recognized by and interacts with the enzyme, enabling effective analyte detection via EEB devices.
Herein, we discuss EEBs for the detection of biomarkers related to human health status.To date, a myriad of research studies have reported the development of EEBs for the detection of environmental toxins (e.g., pesticides and drinking water contaminants) and food safety testing, 17,18 which impact human health.In this context, Kavetskyy and co-workers recently reported nanomaterial EEB modifications for the detection of phenols in wastewater samples.Produced via the manufacturing of coal tar, resins, plastics, fibers, and other industrial materials, phenolic compounds are commonly found in wastewater effluent and harbor toxicity to human cells.Therefore, quantification of phenolic compounds is critical for human health.The biosensor design for phenol detection featured sulfur-modified titanium oxide nanoparticles to improve the efficiency of laccase function as a binding site on the electrode surface.The phenol sensor produced impressive analytical figures of merit, with a linear dynamic range (LDR) of 10-400 μM, and an LOD of 5 μM. 19In a recent review article from the Sosa-Hernández Research Group, advances in EEBs for the quantification of environmental contaminants (e.g., heavy metals, pesticides, and pharmaceuticals) are discussed, focusing on LODs, stability, and other advantages offered by each of the highlighted sensor designs. 20,21In a recent study relevant to the food and beverage industry, Giménez-Gómez et al. developed an EEB device for monitoring the wine fermentation process to ensure the long-term stability of the beverage products.This biosensor design utilized lactate oxidase (LOx) and horseradish peroxidase (HRP) enzymes to detect lactic acid.The enzymes were functionalized on gold electrode surfaces in a polypyrrole membrane.The use of this enzymatic membrane increased sensor stability, as the device maintained 90% of its initial sensitivity value after 40 days of use.The LOx/HRP EEB device reported an LDR of 1-100 μM with an LOD of 0.52 μM.Their results were validated by comparison of EEB measurements to standard colorimetric methods. 20EEB development for the quantification of environmental pollutants and food preservatives is essential to human health.While these studies report acceptable analytical figures of merit and analyte selectivity, further research is necessary to improve long-term EEB stability.
In terms of direct healthcare applications, EEB technologies have been recently developed for the detection of several biomarkers related to human health and disease status.3][24] Significant improvements in EEB performance for glucose detection have been reported in recent research studies.For instance, the Lokar Research Group developed an EEB device utilizing the pyrroloquinoline quinone-glucose dehydrogenase enzyme (PQQ-GdhB) as a surface modification for glucose selectivity in complex biological matrices (Figs.1B-1C). 25To attach the functional enzyme, the electrode surface was further modified with self-assembled monolayers (SAMs) of 6-mercaptohexanol (6-MCH) and 11-mercaptoundecanoic acid (11-MUA).The interactions of the thiolated ends of these molecules with the gold electrode resulted in improved enzyme stability.This sensing device employed ferrocene methanol (FcMeOH) as an electron mediator. 26The reported LOD was 30 nM with an LDR of 30-200 nM.Additionally, this EEB implemented microfluidic engineering enabling continuous glucose measurements. 25In another study of EEB development for glucose sensing, Jin and co-workers demonstrated the use of a mixture of poly(N-phenyl glycine) (PPG) and glucose oxygenase (GOx) as the binding substrate and enzyme, respectively. 27The carbon electrode surface was modified with Prussian blue as the electron transfer mediator, in addition to a polyurethane film serving as a permeable membrane to limit cytotoxicity.This EEB design achieved a wider LDR of 1-30 mM, yet lower sensitivity compared to the Lokar biosensor device.However, the LOD was not reported for this EEB platform, 27 which should be determined in future work.
In addition to EEB glucose detection, enzyme-based EBs have been reported for the quantification of other human health biomarkers, including lactate and cholesterol.For example, the Cheng Research Group recently developed a wearable EEB for lactate detection in human sweat via selective binding to lactase oxidase enzyme immobilized on electrode surfaces.This EEB implemented a dry-spinning method to fabricate stretchable gold fibers as the working electrode to enable wearability on human test subjects.This device achieved an LOD of 0.137 mM, adequate for the average lactate concentration in human sweat (∼6 mM). 28Although this functional EB design presents several advantages in analytical biomarker detection, the sensor stability was only tested at 15 min intervals, rather than over several days or weeks. 29Additionally, Wu and co-workers reported an EEB for the quantification of cholesterol in human blood.This biosensor employed cholesterol oxidase to selectively detect the analyte on graphene-quantum-dot-modified glassy carbon electrodes.Furthermore, the authors utilized cerasome membranes to further modify the electrode surfaces, improving sensor selectivity in complex biological matrices.The authors reported a clinically relevant LDR ranging from 16.0 μM to 6.2 mM and an LOD of 5.0 μM for cholesterol detection in buffered solutions.While this study discusses several useful electrode modifications for sensing applications, the EEB device was not tested for endogenous cholesterol levels in human biofluids over a long-term period, which is essential to future healthcare applications. 30These studies on EEB development for the detection of health biomarkers indicate the need for further research in prolonged sensor use, specifically with regard to modifications and improvements of enzyme structure and electrode immobilization techniques for the enzyme-based recognition element to improve EEB stability.
Research studies focusing on EEBs for health applications have addressed limitations in analytical biosensor performance.Advancements in electrode material modifications, as well as the utilization of novel enzymes, continue to enhance analytical figures of merit in these devices and expand detection capabilities to different types of biomarkers beyond glucose.However, challenges remain in the current EEB design for practical applications.Namely, enzyme stability is a crucial factor in the rational design of EEBs, specifically in terms of pH and temperature independence for sampling in biological fluids.Establishing immobilization methods to improve enzyme stability on the electrode surface is critical for maintaining consistent, long-term current signals, which is essential for continuous, in situ monitoring of health-related biomarkers. 10dditionally, further electrode surface modifications for enzyme immobilization and genetic engineering of enzymes are necessary to design EEB with enhanced stability for sensing in complex biological matrices with interfering species (e.g., ascorbic acid, dopamine, and uric acid). 27Furthermore, future work should focus on evaluating and improving EEB electrode materials toward practical sensing, encompassing long-term enzyme stability and large-scale production capabilities. 9crobial electrochemical biosensors for health monitoring.-Microbialelectrochemical biosensors (MEBs) implement microorganisms as the bioreceptor element, 1 which exhibits several properties that are advantageous for EB applications.These include (1) adaptability to different environments, 31 (2) long-term stability for continuous sensing, and (3) multi-analyte detection via genetic engineering of microorganisms.32 These beneficial properties are also associated with limitations, including lower selectivity and longer response times.33,34 In MEBs, the microbial recognition element is immobilized onto an electrode surface.Effective microbe immobilization is essential, as it contributes to higher biosensor stability and reusability. Taditional bacterial immobilization methods include surface adsorption, encapsulation, entrapment, covalent binding, and cross-linking, which are dependent on electrode material and geometry.4,35 Adhesion of the microorganism on the electrode surface enables electron transfer processes to occur when potential is applied to the working electrode.MEBs encompass one of two general electron transfer mechanisms (Fig. 1D): (1) direct electron transfer where electrode surfaces are in contact with membrane-bound redox-active proteins or (2) mediated electron transfer via endogenous (e.g., flavins, quinones, phenazines) or exogenous (e.g., methylene blue) redox-active mediators facilitating electron transfer at the microbe-electrode interface. 36The first reported MEB implemented Acetobacter xylinum as the recognition element for the measurement of ethanol oxidation, according to ECS Sensors Plus, 2024 3 020603 remarks from Divies in 1975.37 Since this development, several MEBs have been reported encompassing a variety of microbes, as well as electrode materials and modifications for applications in environmental pollution, food safety, and healthcare, which are all relevant to human health. 1 Recent research studies on MEB designs have implemented a variety of microorganisms, including Chlorella, Circinella, Porphyridium cruentum, Rhizopus arrhizus, and Pseudomonas aeruginosa. 1 These bacteria, among others, interact with the analyte of interest to produce an electrochemical signal for the detection of heavy metals, water contaminants, ethanol, caffeine, glucose, food additives, pharmaceuticals, and other biomolecules relevant to health monitoring.1,38 For instance, a recent study from Li et al. reported the development of an MEB for glucose monitoring in human urine for diabetes management.This device addresses the limitations of current glucose-detection technologies by displaying reusability and cost-effectiveness.The design encompassed cylinder-sensor electrodes comprised of stainless-steel mesh and graphite-modified titanium wire.Human urine samples were tested, achieving a response time of 100 s and an LDR of 0.3-5 mM. Th study did not report LOD for this EB device, necessitating further characterization of the analytical figures of merit.The reported analytical performance of the MEB was comparable to commercial glucose meters. 39 Inanother study by Zaib and co-workers, an MEB was developed for the detection of As(III) in contaminated drinking water sources.This sensor utilized carbon paste electrodes modified with Porphyridium cruentum as the bioreceptor.The device reported an LDR of 2.5-20 μg l −1 and an LOD of 1.08 μg l −1 .Additionally, in the presence of low concentrations of other metal ionic species, the MEB maintained a consistent analyte signal.However, as the concentration of the interfering compounds was increased, the signal intensity was affected, revealing limitations in sensor selectivity.40 Another study from the Chee Research Group reported the MEB design for the quantification of trichloroethylene (TCE) in soil and groundwater.TCE is leached into the environment from use in industrial processes and dry cleaning, and is toxic to humans, impacting the nervous system upon exposure.The MEB in this study employed Pseudomonas putida to detect TCE by degrading the contaminant into toluene as a source of chloride ions.The ions were then detected by an ion-selective chloride electrode, producing a current signal directly proportional to TCE concentration. The evice had an LOD of 0.05 mg l −1 with an LDR of 0.05-4 mg l −1 .41 The sensor proved to deliver a consistent analyte signal over five days, indicating good stability.However, this device displayed longer response times of 10 min, limiting real-time measurements.Overall, the discussed studies of MEB designs for health monitoring achieved viable stability over several days.However, these devices were limited in selectivity and response time for trace analyte detection.
The use of microorganisms as the recognition element in EBs is advantageous for stability, reusability, cost-effectiveness, and multianalyte detection capabilities. 1The advantages of MEBs can be attributed to the adaptability of microbes to various environmental conditions, the simplistic, inexpensive growth methods for bacteria, and the ability to genetically modify the cells for specific substrates. 31,32Although the use of microorganisms in sensor design offers benefits for device performance, MEBs are associated with challenges due to the inherent limitations of bacteria.As revealed in recent MEB development studies for health monitoring, [39][40][41] the sensitivity, response time, and analyte selectivity in complex biological matrices proved to be the limiting factors in the MEB performance.These drawbacks necessitate further research in genetic engineering and bacterial metabolic pathways to enhance MEB selectivity and response times. 34ture Needs and Prospects Surface-display techniques for health biosensing.-Asdescribed, EBs can be divided into two main categories based on the recognition element: (1) affinity biosensors and (2) catalytic biosensors. 42EEBs for health monitoring provide several advantages, including high sensitivity that enables low detection and quantification limits, and high specificity for biological analytes of interest, both of which utilize the enzyme-analyte affinity. 43,44dditionally, enzyme-based biosensors have advantages associated with their practical application as sensing platforms. 454][45][46] However, the main challenges with EEBs are the instability of the response, which is associated with enzyme leaking and deactivation, the short lifetime of enzymes at varying environmental conditions (e.g., temperature, pH levels), and passivation of the conductive electrode surface. 1 Future work in the field of enzyme-based electrochemical sensors necessitates improving the stability of enzymes via bioengineering methods 1 to enable early diagnosis of human diseases.
On the other hand, MEBs developed by immobilization of microorganisms to electrode surfaces offer various advantages over isolated enzymes for biosensing applications, such as (1) lower cost due to the exclusion of time-consuming and costly methodologies required for enzyme extraction and purification, (2) ability to resist pH and temperature changes due to enzyme retention in their natural environment, and (3) capability to monitor enzyme activity. 32While MEBs offer advantages over enzyme-based sensors, they have lower degrees of sensitivity and selectivity. 33,47dditionally, the response of MEBs is relatively longer resulting from the diffusional limitations associated with the bacterial cell membrane. 34,48To address these drawbacks, cellular biology, as well as protein and metabolic engineering approaches have been rapidly advancing the field of EBs. 1,34Specifically, metabolic engineering approaches are being employed to modify microorganisms to express specific proteins of interest to improve the response time and sensitivity of the microbial biosensor.Additionally, these strategies could enhance the microbial biosensor selectivity through either the expression or activation of desired metabolic pathways while also suppressing the non-preferred ones.
As part of EEB and MEB advancements, cell-surface-display technologies can be utilized to surface display enzymes on the surfaces of whole living microbial cells.In this context, the display of enzymes on the surface of microorganisms is becoming a fundamental tool to overcome issues in the rational design of EBs. 6 The surface display of enzymes on living microorganisms allows for rapid access to soluble substrates while also retaining the metabolic potential of intracellular enzyme systems.Surface-display methods combine the advantages of microbial-based biosensing systems with cell culturing and metabolic engineering.Enzymes immobilized on the surface of microbial cells have been shown to have improved stability to environmental changes, such as temperature and pH levels.Additionally, the time-consuming and high-cost isolation and purification of enzymes, as well as enzyme immobilization processes 34 can be eliminated by anchoring enzymes on microbial cell surfaces.Furthermore, while most purified enzymes cannot be reused, 49 microorganisms expressing active enzymes on their cell surfaces could be used over extended periods without significant loss of enzymatic activity and can be used repeatedly. 50or a successful enzyme surface display, it is necessary to choose the appropriate enzyme that is selective for the analyte of interest being detected.Selecting an adequate enzyme is critical because it contains signal sequences for an efficient promotion of transportation, immobilization, and stability to the cell surface. 51Furthermore, a variety of anchoring motifs for protein display at the cell surface are presently available for EB design.A successful enzyme anchoring mechanism should provide a stable surface display via strong binding interaction with the cell wall and efficient protein transport throughout the cell. 52Several effective anchoring methods have been previously reported, 53 including the use of outer cell membrane proteins.Namely, ice nucleation proteins (INPs) are produced by microorganisms that can survive in low-temperature environments.This class of outer membrane proteins is efficient in the transport ECS Sensors Plus, 2024 3 020603 and aggregation of enzymes at the cell membrane surface, deeming INPs an effective anchoring motif for surface-display techniques in whole microbial cells. 54Depending on the anchoring enzyme, the carrier enzyme can be fused with either an N-or a C-terminal.If the anchoring enzyme is located on the C-terminal end, the carrier will be on the N-terminal side, but in cases of anchoring motifs fused Nterminally, the carrier will be on the C-terminal side. 55Affinity proteins are typically the main targets of surface-display methods, and these proteins have the potential to be employed as molecular recognition layers due to the surface-displaying cells acting as a biological matrix from which the target analyte is detected. 56ell surface-display systems have examined a myriad of host organisms with different degrees of complexity, ranging from bacterial spores 57 to eukaryotes 58 to prokaryotes. 59Despite differences in these host microorganisms, cell surface-display systems have three standard core features, namely (1) signal peptides to direct enzyme of interest towards a secretory pathway, (2) endogenous surface enzymes flexible to recombination (e.g., fusion, insertion, deletion) to enable stable surface anchoring of enzyme of interest, and (3) a tag to facilitate the detection of an effective surface display.The choice of the most suitable microorganism system for enzyme surface display depends on several factors, such as the complexity of the recombinant enzyme being displayed and its intended application.Eukaryotic system displays, specifically yeast display, have been widely used in surface-display methods, which offer several advantages, including the ability to fold, process, and present complex enzymes, 60 the availability of an established genetic toolbox, and the capability to tolerate harsh conditions. 61For instance, a recent study has shown the construction of a precisely localized enzyme cascade by the integration of two sequential enzymes, glucoamylase and glucose oxidase, on yeast cell surfaces through an a-agglutinin reception as anchoring motif with cohesindockerin interaction. 624][65][66] While the utilization of yeast for surface display provides advantages, it has certain drawbacks, specifically (1) yeast cell growth is significantly slower than that of most bacteria, and (2) yeast surface display has low transformation efficiency limiting the enzyme mutant library size of directed-evolution-based engineering strategies in yeast.On the other hand, prokaryotic surface-display systems most commonly use E.coli as a host for cell surface display of enzymes due to its well-established genetic toolbox and its ability to achieve highdensity surface display of full-length recombinant proteins.
Given the various advantages outlined above, surface-display methods offer promise as a technology to be implemented in the design of electrochemical sensors in order to address the aforementioned challenges associated with enzymatic and microbial EBs.For example, Zhao and co-workers reported the development of wholecell biosensors fabricated via immobilizing yeast cells on electrodes, for the electrochemical detection of glucose and cholesterol levels in patients suffering from chronic diseases. 65Specifically, the researchers employed surface-display methods to anchor glucose dehydrogenase from Aspergillus oryzae and cholesterol oxidase from Chromobacterium sp. on the surface of Saccharomyces cerevisiae (Fig. 2).The assay times were 8.5 s and 30 s for the glucose and cholesterol EBs, respectively.The electrochemical ECS Sensors Plus, 2024 3 020603 sensor for cholesterol detection had a concentration range of 2-6 mmol l -1 while the glucose biosensor exhibited a linear dynamic range from 1.4 to 33.3 mmol l −1 .Further study is necessary for the characterization of the analytical performance of this EB design specifically with regard to the sensor's sensitivity and LOD.The developed biosensors demonstrated respectable performance at room temperature and promising long-term stability over three weeks.Additional work by Wang et al. has demonstrated the surface display of glucose oxidase on S.cerevisiae as a whole-cell biocatalyst for the electrochemical detection of glucose. 66In this work, the surfacedisplayed glucose oxidase was demonstrated to have high specificity, thermal stability, and pH stability.In this biosensor design, the glucose oxidase surface-displaying cells were immobilized on the surfaces of glassy carbon electrodes for glucose monitoring via cyclic voltammetry.The glucose biosensor displayed a linear response ranging from 0.1 to 14 mM with a detection limit of 0.05 mM and showed good accuracy in real sample measurements.

Conclusions
In conclusion, compared to EEBs and MEBs, surface-display approaches have demonstrated promise for a few EBs related to the selective monitoring of biomarkers or target analytes related to human health.However, future work is necessary to develop and characterize EBs based on surface-display recognition.Specifically, these surface-displaying enzymes on the surface of whole cells need to be meticulously characterized as recognition elements.Additionally, EBs employing surface-display technologies need to be evaluated and optimized for health sensing applications in complex biological samples with regard to the analytical figures of merit, including biosensor response time, selectivity, and sensitivity.Finally, future research needs to focus on developing and implementing surface-display-based EBs for in vivo monitoring and sensing in environments related to human health.

Figure 1 .
Figure 1.(A) Mechanisms of direct electron transfer and indirect electron transfer in third-generation and second-generation EEB designs, respectively.Reprinted with permission from Ref. 26.Copyright 2023 from MDPI.(B) Design of PQQ-GdhB EEB integrated with microfluidics device for glucose detection.Working and counter electrodes are Au-based, while the reference electrode is Ag/AgCl.The SAMs interact with the gold surface, creating a stable monolayer.The microfluidic chip is formed, which allows continuous biofluid sampling through covalent enzyme binding.Adapted with permission from Ref. 25.Copyright 2023 from MDPI.(C) PQQ-GdhB reaction with redox mediator FcMeOH at the gold electrode-buffer solution interface.Adapted with permission from Ref. 25.Copyright 2023 from MDPI.(D) Electron transfer mechanisms in MEBs at the microbe-electrode surface interface, including direct electron transfer and mediated electron transfer.Adapted with permission from Ref. 36.Copyright 2021 from MDPI.

Figure 2 .
Figure 2. Overview of glucose dehydrogenase and glucose oxidase surface-display for electrochemical biosensing of glucose and cholesterol.(A) Vector map where enzymes glucose dehydrogenase (Gdh1) or cholesterol oxidase (Cho1) (in blue) are expressed C-terminally.(B) Surface display of Gdh1 and Cho1 on yeast cells.(C) Whole-cell EB design by yeast surface-display cell immobilization.(D) Portable electrochemical monitor for the detection of glucose and cholesterol.Adapted with permission from Ref. 65.Copyright 2020 from MDPI.