Many beneficial proteins have limited natural availability, which often restricts their supply and thereby reduces their potential for therapeutic or industrial usage. The advent of recombinant DNA (rDNA) technology enables the utilization of different microbes as surrogate hosts to facilitate the production of these proteins. This microbial technology continues to evolve and integrate with modern innovations to develop more effective approaches for increasing the production of recombinant biopharmaceuticals. These strategies encompass fermentation technology, metabolic engineering, the deployment of strong promoters, novel vector elements such as inducers and enhancers, protein tags, secretion signals, synthetic biology, high-throughput devices for cloning, and process screening. This appraisal commences with a general overview regarding the manufacture of recombinant proteins by microbes and the production of biopharmaceuticals, their trends towards the development of biopharmaceuticals, and then discusses the approaches adopted for accomplishing this. The design of the upstream process, which also involves host selection, vector design, and promoter design, is a crucial component of production strategies. On the other hand, the downstream process focuses on extraction and purification techniques. Additionally, the review covers the most modern tools and resources, methods for overcoming low expression, the cost of producing biopharmaceuticals in microbes, and readily available recombinant protein products.

The present study aimed to develop an aptamer-based FRET detection strategy for the specific and sensitive detection of AFB1 in contaminated food grains. The study comprises generation of ssDNA aptamers against AFB1 by whole-cell SELEX and their application in a FRET-based platform utilizing graphene oxide (GO) and quantum dots (QDs). The generated aptamers were characterized to determine their specificity and sensitivity using indirect ELISA where AFB1–OVA was used as a coating antigen. Among the aptamers generated, the ATB1 aptamer showed good reactivity and selectivity against AFB1. This aptamer was further characterized to determine its secondary structure and KD value, which was found to be 5.9 kcal mol⁻¹. The characterized aptamers were conjugated onto Cd/Se quantum dots to develop a fluorimetric system for the detection of aflatoxin B1 using a graphene oxide platform. The presence of graphene oxide quenches the fluorescence ability of the quantum dots due to π–π stacking interactions between the aptamer and GO. Upon target addition, the aptamer forms a complex with aflatoxin B1 thereby restoring the fluorescence intensity. The developed assay shows a linear response from 0.002 μg μl⁻¹ to 0.2 μg μl⁻¹ with a detection limit of 0.004 μg μl⁻¹ for the AFB1 standard toxin and showed no cross-reactivity with other closely related mycotoxins. To validate the reliability of the developed method, several field samples spiked with AFB1 were included in this study and the results obtained were cross verified using a standard commercial AFB1 kit. In conclusion, the developed method may find good utility in routine food testing laboratories for risk assessment of AFB1.

The speedy analysis of food products remains a keen area of concern; thus, rapid, highly efficient and robust on-site detection platforms are essential. Globally, Escherichia coli O157:H7 is the most common organism associated with food borne illness. The present study focuses on the development of a rapid onsite E. coli O157:H7 detection platform. Accordingly, target specific ssDNA aptamers are generated using whole cell-SELEX, where the highly reactive aptamers (EcoR1 and EcoR2) with the lowest dissociation constants (K_d) are further biotin tagged (EcoR1) and fluorescent labelled (EcoR2). Subsequently, the biotin-labelled aptamers are immobilized onto silane-glutaraldehyde-functionalized glass slides, which act as capturing ligands, and the QD-labelled aptamers act as revealing probes. The detection limit of the platform is found to be 10² CFU mL⁻¹ within 15 min, and no cross-reactivity towards other related pathogens is observed. The developed assay is further evaluated in complex food matrices to determine the presence of E. coli O157:H7, where the results are found to be consistent with those of the conventional methods with a recovery percentage ranging from 76% to 91%. In conclusion, the developed method is promising for application in real-time food sample monitoring.

The present study was aimed to develop a novel antibody-aptamer based hybrid detection strategy for specific and sensitive detection of aflatoxin B1 (AFB1) from contaminated food grains. The study comprises generation of ssDNA aptamers and anti-AFB1 IgG against AFB1 toxin. The generated bio-probes (aptamers and antibodies) were further characterized for their specificity and sensitivity using indirect ELISA. The generated aptamers namely AFB1a and AFB1b showed prominent reactivity and selectivity against AFB1 toxin. These aptamers were further characterized for their secondary structures and dG values were determined as −4.6 and −2.75 Kcal/mol, respectively. The detection limit (LOD) of AFB1a and anti-AFB1 IgG was determined as 5 and 10 ng/mL, respectively. The characterized aptamers and antibodies against AFB1 were used to develop the sandwich immunoassay. Anti AFB1 IgG was used as a capturing antibody whereas anti-AFB1a aptamer was used as its revealing partner in the assay. The limit of detection (LOD) of the immunoassay was determined to be 5 ng/mL of AFB1 standard toxin and showed no cross-reactivity with closely related mycotoxins. To assess the reliability of the developed method, several field samples contaminated with aflatoxin B1 was included in the study and results were validated with commercial AFB1-ELISA Kit. Additionally, the spiking studies were also carried out to demonstrate the consistency and dependability of the developed hybrid sandwich immunoassay wherein the toxins recovered were found to be ranging between 73 and 98.80% with the LOD at 5 ng/mL. In conclusion, the developed method may find the better utility in routine food testing laboratories for assessment of AFB1.

The present study involves immunoassay platform development based on a surface functionalized silica matrix for rapid onsite detection of Staphylococcal enterotoxin B (SEB). Silica matrix functionalization as well as the immunoassay parameters was experimentally designed and optimized through response surface methodology (RSM). Silica surface functionalization was carried out with hydrofluoric acid (HF), ammonia, 3-aminopropyl triethoxysilane (APTES) and glutaraldehyde (GA). The RSM optimized matrix functionalization parameters for HF, ammonia, APTES and GA were determined to be 10%, 40%, 20% and 10% (V/V), respectively. Antibodies for the study were generated against recombinant SEB toxin in rabbit (anti-SEB IgG) and chicken (anti-SEB IgY). Subsequently, antibodies were immobilized on the functionalized silica matrix and were further characterized by SEM and contact angle measurements to elucidate the surface uniformity and degree of hydrophilicity. The immunoassay platform was developed with anti-SEB IgG (capturing agent) and anti-SEB IgY (revealing partner). The limit of detection (LOD) of the developed platform was determined to be 0.005 μg mL⁻¹ and no cross-reactivity with similar toxins was observed. Upon co-evaluation with a standard ELISA kit (Chondrex, Inc) against various field isolates, the platform was found to be on par and reliable. In conclusion, the developed method may find better utility in onsite detection of SEB from resource-poor settings.

Natural biodegradable polymers serve as a more sustainable and cost effective solution for food packaging compared to synthetic alternatives.