The interaction between nanoparticles and proteins is a central problem in the nano-bio-fields. However, it is still a great challenge to characterize the specific interaction between nanoparticles and proteins in structural details. Using the Goldbodies, the artificial antibodies created by grafting complementary-determining regions (CDRs) of natural antibodies onto gold nanoparticles, as the models, we manage to identify the key residues of the CDR peptides on gold nanoparticles for the specific interactions by alanine scanning mutagenesis. Each and every residue of the CDR peptides on two Goldbodies (which specifically bind with hen egg white lysozyme and epidermal growth factor receptor, respectively) is mutated to alanine one by one, generating a total of 18 single-mutants of the two Goldbodies. Experimental results reveal that the key residues of the CDR peptides for the specific interactions between the two Goldbodies and the corresponding antigens are exactly the same as those in the natural antibodies, thus proving that the correct conformations of the CDRs of natural antibodies have been successfully reconstructed on AuNPs. This is the first residue-resolution structural illustration for the specific interaction between a designed nanoparticle and a protein.
Many efforts have been made to develop inhibitors of MDM2 as potential drugs for cancer therapy. In this work, we use our previous developed conformational engineering technique to stabilize the binding conformation of the p53 transcription activation domain (TAD) peptide on gold nanoparticles (AuNPs), and create an AuNP‐based anti‐MDM2 artificial antibody, denoted as anti‐MDM2 Goldbody, that specifically binds MDM2. Though the free TAD peptide is unstructured, circular dichroism (CD) spectra confirm that its α‐helical conformation in the original p53 protein is restored on the anti‐MDM2 Goldbody, and surface plasmon resonance (SPR) experiments confirm that there is strong specific interaction between the anti‐MDM2 Goldbody and MDM2, demonstrating the anti‐MDM2 Goldbody as a potential inhibitor of MDM2. This work demonstrates that the conformational engineering technique is not limited to the antigen‐antibody systems, but can also be applied more widely in other protein‐protein interfaces to create increasingly more artificial proteins for various biomedical applications.
It
is generally believed that a protein’s sequence solely
determines its native structure, but how the long- and short-range
interactions jointly determine the native structure/conformation of
the protein or every local fragment of the protein is still not fully
understood. Since most protein fragments are unstructured on their
own, direct observation of the folding of flexible protein fragments
is very difficult. Interestingly, we show that it is possible to graft
the complementary-determining regions (CDRs) of antibodies onto the
surface of a gold nanoparticle (AuNP) to create AuNP-based artificial
antibodies (denoted as Goldbodies), such as an antilysozyme Goldbody.
Goldbodies can specifically recognize the corresponding antigens like
the original natural antibodies do, but direct structural evidence
for the refolding or restoration of native conformation of the grafted
CDRs on AuNPs is still missing and in high demand. Herein we design
a new Goldbody that targets an epitope on the lysozyme different from
that of the previous antilysozyme Goldbody, and the one circle of
helix in the CDR makes it possible to distinguish the unfolded conformation
of the free CDR and its folded conformation on AuNPs by circular dichroism
(CD) spectroscopy. The refolding of flexible protein fragments on
NPs provides unique evidence and inspiration for understanding the
fundamental principles of protein folding.
Antibody-based immunoassays such as the lateral flow
immunoassay
(LFIA) and enzyme-linked immunosorbent assay (ELISA) are currently
indispensable analytical methods widely used in many fields, mainly
due to the high sensitivity and specificity of antibodies. However,
the high cost of monoclonal antibodies and their susceptibility to
high temperature limit the application of immunoassays. Previously,
we developed a class of gold nanoparticle (AuNP)-based artificial
antibodies, called Goldbody. Goldbodies not only bind antigens as
specifically as monoclonal antibodies do but also have far better
stability than monoclonal antibodies. To take advantage of the excellent
specificity, stability, and easy functionalization of Goldbodies and
use them for the substitution of monoclonal antibodies in immunoassays,
herein, we synthesize a biotinylated anti-lysozyme Goldbody and successfully
construct a competitive LFIA for the detection of lysozyme in the
range of 17.98–226.49 ng·mL–1. At the
same time, the biotinylated anti-lysozyme Goldbody can easily replace
the detection antibody in the commercial BA (biotinylated antibody)-ELISA
kit for the detection of lysozyme with a lower detection limit of
0.34 ng·mL–1 and a wider detection range of
0.89–20 ng·mL–1 compared with the commercial
BA-ELISA kit. In addition, the biotinylated anti-lysozyme Goldbody
has good thermal stability in both assays and can accurately detect
spiked samples even after pretreatment at 100 °C, demonstrating
the high potential of Goldbodies as a good replacement of monoclonal
antibodies in immunoassays.
Cross-border e-commerce is also facing the problems of cross-border goods selling counterfeit, long delivery time of cross-border logistics, and high cross-border payment fee in the process of development. Blockchain technology, with its characteristics of high trustworthiness, traceability, and information immutability, can be applied to the field of cross-border e-commerce, providing a new direction for cross-border e-commerce to solve these problems. At the present stage, scholars at home and abroad mainly study how blockchain technology is applied in cross-border e-commerce, explore the specific mode of combining blockchain technology with cross-border e-commerce, and lay the theoretical foundation for the application of blockchain technology in cross-border e-commerce, while less research is conducted on the application effect of blockchain technology in cross-border e-commerce. Based on this, this paper researches the influence mechanism of the application of blockchain technology in cross-border e-commerce on consumers’ willingness to purchase, explains the current situation of the application of blockchain technology in various fields of cross-border e-commerce, and on this basis, divides the quality of the blockchain system of cross-border e-commerce into three dimensions of commodity information quality, logistics service quality, and payment security.
The flocculation of small surficial groups on pristine CNCs (carbon nanocoils) bundles limit their application. In this study, we designed and fabricated novel array CNCs with a surficial decoration of polyaniline (PANI) using in situ methods. Atomic layer deposition (ALD) and chemical vapor deposition (CVD) methods were employed to fabricate the highly pure array CNCs. The array CNCs decorated with ultra-thin PANI were confirmed by different characterizations. Furthermore, this material displayed a good performance in its detection of formaldehyde. The detection results showed that the CNCs coated with PANI had a low limit of detection of HCHO, as low as 500 ppb, and the sensor also showed good selectivity for other interfering gases, as well as good repeatability over many tests. Furthermore, after increasing the PANI loading on the surface of the CNCs, their detection performance exhibited a typical volcanic curve, and the value of the enthalpy was extracted by using the temperature-varying micro-gravimetric method during the process of detection of the formaldehyde molecules on the CNCs. The use of array CNCs with surficial decoration offers a novel method for the application of CNCs and could be extended to other applications, such as catalysts and energy conversion.
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