Electrochemical Response of Redox Amino Acid Encoded Fluorescence Protein for Hydroxychloroquine Sensing.

The sudden rise in the demand has led to large-scale production of hydroxychloroquine (HCQ) in the global market for various diseases such as malaria, rheumatic arthritis, and systemic lupus erythematous and prophylactic treatment of early SARS-CoV-2 outbreak. Thorough monitoring of HCQ intake patients is in high demand; hence, we have developed a redox amino acid encoded fluorescent protein-based electrochemical biosensor for sensitive and selective detection of HCQ. This electrochemical biosensor is generated based on the two-electron transfer process between redox amino acid (3,4-dihydroxy-L-phenylalanine, DOPA) encoded bio-redox protein and the HCQ forms the conjugate. The DOPA residue in the bio-redox protein specifically binds with HCQ, thereby producing a remarkable electrochemical response on the glassy carbon electrode. Experimental results show that the developed biosensor selectively and sensitively detects the HCQ in spiked urine samples. The reagent-free bio-redox capacitor detects HCQ in the range of 90 nM to 4.4 µM in a solution with a detection limit of 58 nM, signal to noise ratio of 3:1, and strong anti-interference ability. Real-time screening, quantification, and relative mean recoveries of HCQ on spiked urine samples were monitored through electron shuttling using bio-redox protein and were found to be 97 to 101%. Overall, the developed bio-redox protein-based sensor has specificity, selectivity, reproducibility, and sensitivity making it potentially attractive for the sensing of HCQ and also applicable to clinical research.

Engineering microbial cells with metal chelating hydroxylated unnatural amino acids for removable of synthetic pollutants from water.

Lead (Pb2+) is a well-known heavy metal and toxic synthetic industrial pollutant in the ecosystem and causes severe threats to living organisms. It is paramount to develop a sustainable microbial engineering approach to remove synthetic pollutants from the environment. Genetic code engineering is emerging as an important microbial engineering tool in biosciences to biosynthesis congener protein production beyond the canonical set of natural molecules and expand the chemistries of living cells. Here, we prepare cells expressing unnatural amino acid encoded congener proteins for effectively removable toxic synthetic industrial pollutants (Pb2+) with high binding efficiency. Native and the developed congener proteins expressing cells adapted the Langmuir and Sips adsorption model that recommends uniform adsorption with Pb2+ ions. This could be due to a more significant number of functional groups on the protein surface. Fluorescence spectroscopic, field emission scanning electron microscope, X-ray photoelectron spectroscopic analysis, and protein-metal molecular stimulation coordination allowed us to explore the role of hydroxylation on Pb2+ adsorption. The bioreactor filled with immobilized protein-containing active granules showed >90% of lead removal in the contaminated water samples. The desorption of bound Pb2+ from GFP and its variants were studied by varying the pH to reuse the proteins for subsequent usage. We observed that about 70% of the GFP and its variants could be recycled and >75% of fluorescence efficiency could be recovered. Among all the variants, GFPHPDP exhibits high affinity and maintains the reusability efficiency in 7 consecutive cycles. These results suggest that genetic code engineering of cells encoding unnatural amino acids could be a next-generation microbial engineering tool for manipulating and developing the microbial strain's selective and effective removal of synthetic pollutants from the environment.

Non-proteinogenic amino acid based supramolecular hydrogel material for enhanced cell proliferation.

Supramolecular gel material built from low-molecular-weight (LMW) gelators finds potential applications in various fields, especially in drug delivery, cell encapsulation and delivery, and tissue engineering. The majority of the LMW gelators in these applications are based on functionalized peptides/amino acids consisting of proteinogenic amino acids which are proteolytically unstable. Herein, we have developed a new LMW gelator containing non-proteinogenic amino acid namely 2,3-diaminopropionic acid (Dap), a key precursor in the synthesis of many antibiotics namely viomycin and capreomycin, by functionalizing with fluorenylmethoxycarbonyl at both amino terminals of Dap [Fm-Dap(Fm)]. Hydrogelation test at different pH indicates that Fm-Dap(Fm) can form a hydrogel in a wide range of pH (4.9 to 9.1) with minimum hydrogelation concentration depends on the pH. The mechanical strength and thermal stability of the Fm-Dap(Fm) hydrogel material are found to decrease with increasing pH (acidic > neural/physiological > basic). The thermal stability of Fm-Dap(Fm) hydrogels is pH-dependent and elicits high stability at acidic pH. Also, Fm-Dap(Fm) hydrogels exhibit strong thixotropic property where regelation (self-healing) occurs upon release of stress. Morphological analysis indicates the formation of fibrils, which are entangled to form three dimensional network structures. Several spectroscopic measurements provided evidence for the self-assembly of Fm-Dap(Fm) molecules through intermolecular aromatic π-π stacking and hydrogen bonding interactions during hydrogelation. Interestingly, Fm-Dap(Fm) not only exhibits hydrogel formation but also shows cell viability and enhanced cell proliferation at physiological pH (7.4). Further, Fm-Dap(Fm) forms a hydrogel upon co-incubation with vitamin B12 and also exhibits release of vitamin B12 over a period. The current study thus demonstrates the development of a new hydrogel material, based on LMW gelator containing the non-proteinogenic amino acid, which can elicit cell viability, enhanced cell proliferation, drug encapsulation, and drug release properties. Hence, Fm-Dap(Fm) hydrogel could be an ideal material for biomedical applications, especially in tissue engineering and drug delivery.

A self-assembly and higher order structure forming triple helical protein as a novel biomaterial for cell proliferation.

Collagen plays a critical role in the structural design of the extracellular matrix (ECM) and cell signaling in mammals, which makes it one of the most promising biomaterials with versatile applications. However, there is considerable concern regarding the purity and predictability of the product performance. At present, it is mainly derived as a mixture of collagen (different types) from animal tissues, where the selective enrichment of a particular type of collagen is generally difficult and expensive. Collagen derived from bovine sources poses the risk of transmitting diseases and can cause adverse immunologic and inflammatory responses. Hence, recombinant collagen can be a good alternative. Nevertheless, the necessity of post-translational hydroxyproline (Hyp) modification limits large-scale recombinant collagen production. Here, we recombinantly expressed the collagen-like protein (CLTP) and genetically introduced the Hyp in the CLTP to form a higher order self-assembled fibril structure, similar to human collagen. During the current study, it was observed that the Hyp incorporated CLTP protein (CLTHP) formed a stable triple helical polyproline-II like structure and self-assembled to form fibrils at neutral pH, which had an initial lag phase followed by a growth phase similar to animal collagen. In contrast, the higher order fibrillar assembly was missing in the nonhydroxylated CLTP. This study demonstrated that CLTHP self-association is based on the common underlying lateral interactions between triple helical structured proteins, where the hydroxyproline forms the significantly stable hydration network. Hence, this work will be the first fundamental empirical research for flexible modifications of recombinant collagen for structural analysis and biomedical applications.