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Background:The mechanism of heat-induced irreversible denaturation of a single domain VHH camelid antibody remains unclear.Results: The denaturation of VHH did not depend on the number of refolding/unfolding reactions or protein concentration. Mutations replacing Asn increased the heat tolerance of VHH. Conclusion: Chemical modifications are suggested to be a dominant factor for irreversible denaturation of VHH. Significance: Our findings enable further improvements for heat tolerance of VHH.

There is currently substantial interest in creating bioelectronic devices that can be implanted in and attached to humans for sensing and control of organs and internal systems in order to prolong and improve the quality of life.[1-4] Herein we report a new type of implantable bioelectronic device that is operated by laser irradiation from outside the body. Carbon nanotubes (CNTs), which absorb laser light and transform it to thermal energy with high efficiency, are key in the design of this device; the thermal energy is further converted into electrical power with a Seebeck device. The CNT-based photo-thermal-electrical (PTE) converter is unique and has potential in practical use as it can be simply operated by laser irradiation, and can be easily removed or replaced as it is embedded near the skin.Among several photothermally active nanomaterials, CNTs are of particular interest because of their extraordinarily high efficiency of photothermal energy conversion and high light-absorption cross-section in a wavelength range that can be transmitted through living tissue (diagnostic window: 650-1100 nm).[5] The photothermal energy conversion of CNTs is effective in various biological applications. [6][7][8][9][10][11][12][13][14][15][16][17][18][19] It is well known that gold nanoparticles also have photothermal properties, but CNTs can be more effectively heated at various wavelengths because of their strong absorbance over a wide wavelength range.[6] Thermal energy can be converted into electricity by using thermoelectric (TE) conversion devices that utilize the Seebeck effect. [18][19][20] A PTE energy conversion system that combines TE elements and CNTs is available and can be used in the diagnostic window of living tissues. PTE converters are promising devices for electrical power supply to embedded implantable medical devices. We recently fabricated a PTE converter using CNT gels exposed to 1064 nm laser light, and demonstrated that it generated sufficient electrical energy to power a motor and lightemitting diodes. [18,19] However, the applications of our previous PTE converters were limited to ex vivo electrical power generation. Herein, we demonstrate that 1) a well-dispersed single-walled CNT/poly(dimethylsiloxane) (SWNT-PDMS) composite can be formed, and a novel PTE converter fabricated with a CNT polymer composite can effectively convert the photothermal energy of CNTs into electricity; 2) the PTE converter can supply electrical power for stimulation of electrical activity in physiological tissues, such as a heart muscle of zebrafish (Danio rerio) and a sciatic nerve of a frog (Xenopus laevis); and 3) the PTE converter was effective in the body of a rat. These results dramatically extend the concept of a PTE converter into new areas and applications.To prepare the CNT-based PTE converter, we first prepared CNT polymer sheets. We wrapped noncovalent SWNTs with poly(3-hexylthiophene) (P3HT) and dispersed them in PDMS sheets (P3HT-SWNT-PDMS; see Methods in the Supporting Information). The strong tendency of...

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The antigen-binding domain of camelid dimeric heavy chain antibodies, known as VHH or Nanobody, has much potential in pharmaceutical and industrial applications. To establish the isolation process of antigen-specific VHH, a VHH phage library was constructed with a diversity of 8.4 × 10(7) from cDNA of peripheral blood mononuclear cells of an alpaca (Lama pacos) immunized with a fragment of IZUMO1 (IZUMO1PFF) as a model antigen. By conventional biopanning, 13 antigen-specific VHHs were isolated. The amino acid sequences of these VHHs, designated as N-group VHHs, were very similar to each other (>93% identity). To find more diverse antibodies, we performed high-throughput sequencing (HTS) of VHH genes. By comparing the frequencies of each sequence between before and after biopanning, we found the sequences whose frequencies were increased by biopanning. The top 100 sequences of them were supplied for phylogenic tree analysis. In total 75% of them belonged to N-group VHHs, but the other were phylogenically apart from N-group VHHs (Non N-group). Two of three VHHs selected from non N-group VHHs showed sufficient antigen binding ability. These results suggested that biopanning followed by HTS provided a useful method for finding minor and diverse antigen-specific clones that could not be identified by conventional biopanning.