Proteins In Aqueous Solution Research Articles

Aggregation selectivity of amyloid β1‐11 peptide in aqueous ionic liquid solutions

AbstractUnderstanding the aggregation selectivity of peptide fragments of full‐length proteins in aqueous solutions with ionic liquids (ILs) could facilitate the elucidation of the relationship between the IL‐protein interactions and structural behavior of intrinsically disordered proteins (IDPs) such as amyloid β protein following the addition of ILs. In the present study, we investigate structural changes in peptide fragment 1‐11 (Aβ1‐11) of amyloid β protein in aqueous solutions with two ILs including 1‐butyl‐3‐methylimidazolium thiocyanate ([bmim][SCN]) and ethylammonium nitrate (EAN) using optical spectroscopy. The addition of [bmim][SCN], which exhibits strong protein denaturant ability, induced the formation of an intermolecular β‐sheet structure (aggregation), while the addition of EAN, which has a weaker denaturant ability compared with [bmim][SCN], did not cause aggregation. Since the role of cations is related to the ability to mask the charged residues of Aβ1‐11, the aggregation selectivity of Aβ1‐11 depends on the anionic species and anions with high denaturation ability enhanced aggregation. Our results demonstrated that the structural change in peptide fragment in aqueous IL solutions could be used to evaluate the relationship between the IL‐protein interactions and aggregation selectivity in IDPs in aqueous IL solutions.

Advances of the technologies in large-scale membrane proteome identification

Membrane proteins play important functions not only as receptors and transporters, but also in many other important intracellular functions such as photosynthetic and respiratory electron transport. Identification of membrane proteins is a necessary step to understand their functions. Membrane proteins are generally highly hydrophobic and difficult to be resolved by aqueous solutions, and large-scale proteomic identification of membrane proteins has been a great technical challenge. Significant efforts have been invested in the field to improve the solubility of membrane proteins in aqueous solutions that are compatible for mass spectrometry analysis. This review summarizes the main technological achievements in the field of membrane proteomics particularly for the improvement of membrane protein identification, and uses the photosynthetic model cyanobacterium Synechocystis sp. PCC6803 as an example to illustrate how technology advances push forward the field in terms of the increased coverage of membrane proteome identification.

Binding of lanthanide salts to zwitterionic phospholipid micelles

As the use of lanthanide salts in biophysical systems increases and the separation of lanthanides from nuclear and other wastes with extraction processes has become an important technological challenge, a deeper understanding of the behavior of lanthanides at lipid interfaces is urgently needed. In this work the interaction of lanthanide salts with zwitterionic phospholipids is probed using aqueous micelles of the surfactant dodecyl phosphocholine (DPC), which are useful membrane-mimetic model systems, widely used for the solubilization of membrane proteins in aqueous solutions. Because more than one species exists in lanthanide salt solutions, even at the pH value of 4 used in this experiment, the major goal of this investigation is to examine which species are actually binding to the micelles. Using static and time-dependent europium fluorescence, strong indications are obtained that both the Eu3+ cation and its 1:1 chloride, nitrate, or sulfate complexes bind to the micelles, whereas the europium species do not appear to interact strongly with DPC molecules below the cmc. From isothermal titration calorimetry (ITC) measurements it is found that the lanthanide interaction with DPC micelles increases to the right of the lanthanide series and is - surprisingly – endothermic, underlying the important role of hydration effects in the interaction. The anion of the lanthanide salt strongly influences the thermodynamics: perchlorate and sulfate salts give extraordinary results, switching the interaction to exothermic. A multi-level phenomenological electrostatic model of the europium fluorescence lifetimes strongly suggests that in the case of nitrate salts both Ln3+ and LnNO32+ ions bind to the micelles. Overall a detailed molecular picture of the complexity of lanthanide-lipid interactions at interfaces is emerging from these experiments and the associated modelling effort.

Activity and thermal stability of Mycobacterium tuberculosis PE1 and PE2 proteins esterase domain in the presence of aprotic ionic liquids.

An ionic liquid (IL) is a salt in which the ions are poorly coordinated, resulting in these solvents being liquid below 100 °C or even at room temperature. ILs generally consist of large sized anions and cations, have certain unique advantageous properties and hence are considered as 'green solvents'. Thermal stability of the α/β-serine hydrolase (SH) domain in PE1 and PE2 proteins of Mycobacterium tuberculosis (M.tb) possessing esterase activity was studied in the presence of aprotic ILs consisting of imidazolium cations and anions. Addition of ILs to an aqueous solution of proteins prevented their unfolding and aggregation at higher temperatures. The thermal denaturation curve of proteins with ILs shifted to higher temperatures compared to the absence of ILs from CD spectra. The remaining activities of PE1/PE2 proteins with 1.4 M [EMIM][BF4], [EMIM][Cl], [BMIM][BF4] and [BMIM][Cl] exhibited 100%/100%, 58.96%/58.84%, 78.92%/78.94% and 54.63%/54.92% greater activities, respectively after the heat treatment at 30 °C for 35 min. We conclude that the remaining activities of both proteins are sufficiently maintained after the heat treatment and this depends upon the nature, concentration of ILs, and the thermal incubation time. Specifically, [EMIM][BF4] and [BMIM][BF4] exhibit higher thermal stabilization compared to [EMIM][Cl] and [BMIM][Cl].

Protein-protein interactions and water activity coefficients can be used to aid a first excipient choice in protein formulations.

With respect to all biopharmaceuticals marketed to date, monoclonal antibodies represent the largest fraction with more than 48% market share (2012). However, the development of biopharmaceutical formulations is a challenging task, and time-consuming and cost-intensive high-throughput screenings are still state-of-the-art in formulation design. These screening techniques are almost exclusively based on heuristic decisions thus the benefit in terms of mechanistic understanding is often unclear. It requires novel, physical-sound methods to enhance/optimize future formulation development, ideally by understanding molecular interactions in these complex solutions. A suitable and evaluated measure-of-choice to characterize protein-protein interactions in aqueous protein solutions is the second osmotic virial coefficient B22 which can be measured using static light scattering techniques. Furthermore B22 can be modeled/predicted via the extended mxDLVO model for protein-protein interactions in the presence of single excipients and excipient-mixtures. Building up on this approach, giving an additional insight into water-water and water-excipient interactions, the thermodynamic equation-of-state ePC-SAFT is used to calculate water activity coefficients in the presence of excipient-mixtures. Immunoglobulin G (IgG) was chosen as a model protein to predict B22-values for IgG in the presence of model excipient-mixtures (trehalose-NaCl, l-histidine-trehalose, l-histidine-NaCl). The combination of water activity coefficients and B22 allows to quickly identify a first guess on suitable formulation conditions that then can be further evaluated with existing methods/knowledge.

Comparison of Polymer and Ceramic Membrane in the Separation of Proteins in Aqueous Solution Through Liquid Chromatography (Journal of Computational and Theoretical Nanoscience, Vol. 16(1), pp. 157–164 (2019))

Comparison of Polymer and Ceramic Membrane in the Separation of Proteins in Aqueous Solution Through Liquid Chromatography (Journal of Computational and Theoretical Nanoscience, Vol. 16(1), pp. 157–164 (2019))

Plasma protein adsorption on TiO2 nanoparticles: Impact of surface adsorption on temperature-dependent structural changes

Protein adsorption on metal oxide nanoparticle surfaces is crucial to the behavior of oxide nanoparticles in biological systems. In this study, attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used for the first time to probe differences in the temperature-dependent conformational changes of two plasma proteins, bovine serum albumin (BSA) and fibrinogen (Fib), in solution and adsorbed on titanium dioxide nanoparticle (ca. 20 nm in diameter) surfaces. ATR-FTIR spectra and autocorrelation moving window two-dimensional correlation (MW2D) spectroscopy were used to identify the denaturation temperature range of these two proteins in aqueous solution and adsorbed. Generalized two-dimensional correlation spectroscopy (2DCOS) was done to investigate the protein unfolding pathway. The results show that the thermostability of BSA changed when adsorbed on the surface whereas there was no difference for solution phase Fib and adsorbed on TiO2. The most notable fact was that solution BSA underwent significant structural changes upon adsorption whereas adsorbed Fib did not. Upon heating adsorbed BSA, no further changes occurred while BSA in solution thermally denatured. In contrast, solution and adsorbed Fib had identical denaturation temperatures and similar sequential secondary structural changes with increasing temperature. These studies show quantitatively, for the first time, that there are differences in the temperature-dependent structural changes of proteins adsorbed on nanoparticles surfaces and these changes depend on the initial interaction with the nanoparticle surface. Overall, the results of this study provide new insights into understanding the effects of temperature on the structure of adsorbed protein on nanoparticle surfaces and the range of different behaviors that can occur.

Water-insoluble amorphous silk fibroin scaffolds from aqueous solutions.

Regenerated silk fibroin (RSF) is emerging as promising biomaterial for regeneration, drug delivery and optical devices, with continued demand for mild, all-aqueous processes to control microstructure and the performance. Here, temperature control of assembly kinetics was introduced to prepare the water-insoluble scaffolds from neutral aqueous solutions of RSF protein. Higher temperatures were used to accelerate the assembly rate of the silk fibroin protein chains in aqueous solution and during the lyophilization process, resulting in water-insoluble scaffold formation. The scaffolds were mainly composed of amorphous states of the silk fibroin chains, endowing softer mechanical properties. These scaffolds also showed nanofibrous structures, improved cell proliferation in vitro and enhanced neovascularization and tissue regeneration in vivo than previously reported silk fibroin scaffolds. These results suggest utility of silk scaffolds in soft tissue regeneration.

Bovine serum albumin, aqueous solution, by near-ambient pressure XPS

Near-ambient pressure x-ray photoelectron spectroscopy (NAP-XPS) is a less traditional form of XPS that allows samples to be analyzed at relatively high pressures, i.e., at greater than 2500 Pa. With NAP-XPS, XPS can probe moderately volatile liquids, biological samples, porous materials, and/or polymeric materials that outgas significantly. In this submission, we show survey, C 1s, O 1s, and N 1s narrow scans from an aqueous solution of a common protein, bovine serum albumin. The C 1s peak envelope is well fit to four symmetric peaks of equal width that correspond to carbon bonded to carbon and hydrogen (C-1), carbon singly bonded to oxygen (C-2), carbonyl and/or amide carbon (C-3), and carboxyl carbon (C-4). Two possible peak fits are considered for the N 1s and O 1s peak envelopes. The N 1s signal is fit to four peaks that correspond to amine (—NH2), amide (O˭C‒NH2), ammonium (—NH3+), and N2(g) nitrogen, and alternatively to three peaks that correspond to amine, amide, and N2(g) nitrogen. The O 1s peak envelope is similarly fit to three and four components.

Surfactant modulation effect on the fluorescence emission of a dual-fluorophore: Realizing a single discriminative sensor for identifying different proteins in aqueous solutions

A bispyrene-modified perylene derivative (PEPBI), in which the pyrene and the perylenebisimide were connected by a hydrophilic oligoethoxy linker, was designed and synthesized. The absorption spectra and emission spectra revealed that PEPBI has the characteristics of both pyrene and perylene units. The cationic surfactant cetyltrimethylammonium bromide (CTAB) can efficiently regulate the fluorescence emission of PEPBI from pyrene monomer to pyrene monomer-perylene co-emission in aqueous solution. The selected fluorescent ensemble based on PEPBI/CTAB assembly showed ratiometric responses to nonmetalloproteins and turn-off responses to metalloproteins. Collecting the data of fluorescence variation of both pyrene and perylene units at six wavelengths can form specific recognition fingerprints for both nonmetalloproteins and metalloproteins. Using linear discriminant analysis, the sensor ensemble can discriminate four nonmetalloproteins and four metalloproteins. In this work, the combination of surfactant assemblies’ modulation effect and a dual-fluorophore with multiple emission bands realizes the discrimination of same type of proteins by a single sensor system.

Estimation of Shape, Volume, and Dipole Moment of Individual Proteins Freely Transiting a Synthetic Nanopore.

This paper demonstrates that high-bandwidth current recordings in combination with low-noise silicon nitride nanopores make it possible to determine the molecular volume, approximate shape, and dipole moment of single native proteins in solution without the need for labeling, tethering, or other chemical modifications of these proteins. The analysis is based on current modulations caused by the translation and rotation of single proteins through a uniform electric field inside of a nanopore. We applied this technique to nine proteins and show that the measured protein parameters agree well with reference values but only if the nanopore walls were coated with a nonstick fluid lipid bilayer. One potential challenge with this approach is that an untethered protein is able to diffuse laterally while transiting a nanopore, which generates increasingly asymmetric disruptions in the electric field as it approaches the nanopore walls. These "off-axis" effects add an additional noise-like element to the electrical recordings, which can be exacerbated by nonspecific interactions with pore walls that are not coated by a fluid lipid bilayer. We performed finite element simulations to quantify the influence of these effects on subsequent analyses. Examining the size, approximate shape, and dipole moment of unperturbed, native proteins in aqueous solution on a single-molecule level in real time while they translocate through a nanopore may enable applications such as identifying or characterizing proteins in a mixture, or monitoring the assembly or disassembly of transient protein complexes based on their shape, volume, or dipole moment.

The computational and experimental studies on a 1, 2, 3-triazole compound and its special binding to three kinds of blood proteins

A newly synthesized compound, ethyl 5-phenyl-2-(p-tolyl)-2H-1, 2, 3-triazole-4-carboxylate (EPPC) may be considered as a drug candidate and was exploited to study the structural and spectral properties by using quantum chemical calculation and multiple spectroscopic techniques. The results on theoretical spectrum of EPPC were consistent with experimental spectrum in great degree. In addition, EPPC has been as a special probe and investigated on the interactions with three kinds of blood proteins including human serum albumin (HSA), human immunoglobulin (HIgG) and bovine hemoglobin (BHb) by using UV–Vis, fluorescence spectroscopy and molecular modeling, respectively. Changes in various fluorescence and UV–Vis spectra were observed upon ligand binding along with a remarkable degree of fluorescence enhancement on complex formation under physiological condition with binding constant about 105 order of magnitudes, which caused the variations of conformation and microenvironment of these proteins in aqueous solution. The obtained results from the thermodynamic parameters calculated according to the van’t Hoff equation indicated that the entropy change ΔS° and enthalpy change ΔH° were found to be 0.168 KJ/mol K and 22.154 KJ/mol for EPPC-HSA system, 0.284 KJ/mol K and 54.408 KJ/mol for EPPC-HIgG system, and 0.228 KJ/mol K and 37.548 KJ/mol for EPPC-BHb system, respectively, which demonstrated that the primary binding pattern is determined by hydrophobic interaction. The results of docking and molecular dynamics simulation using three proteins crystal models revealed that EPPC could bind to three proteins well into hydrophobic cavity, which showed good consistence with the spectroscopic measurements.Communicated by Ramaswamy H. Sarma

Phytotoxins eliminated by milk: a review

ABSTRACT: Milk is a complex emulsion of lipids suspended in aqueous protein solution that can be a carrier of various contaminants, but generally it is not an important route of toxic excretion. The main problem is chronic repetitive exposure, as it occurs with ingestion of toxic plants and its potential danger to animals that consume the milk. Previously reported hazardous phytotoxins eliminated by milk include: indolizidine alkaloids, causing oligosaccharide storage disease; piperidine alkaloids, causing acute poisoning or malformations; pyrrolizidine alkaloids, which cause hepatic lesions; quinolizidine alkaloids, as a cause of skeletal defects; glucosinolates, which cause changes in the thyroid; tremetol (or tremetone), which causes a disease characterized by tremors in animals and milk sickness in humans; sodium monofluoracetate, which causes the death of kids after ingestion of colostrum from goats that have ingested Amorimia septentrionalis during gestation; ptaquiloside, which induces carcinogenesis in animals that ingest milk or derivatives produced by animals that have ingested Pteridium spp. Ipomoea asarifolia, which contains indole diterpenes causing tremors in suckling pups. Chrysocoma ciliata causes alopecia in suckling pups, but its toxic compound is still unknown. Knowledge about the risk of exposure to these substances via milk and its dissemination are important for veterinary and human health.

Study of Hydration of Biomolecules and Nanoparticles in Aqueous Solutions and Suspensions Using Coherent Laser Spectroscopy

A considerable increase in the concentration of H2O ortho spin isomers and H2O2 molecules in the hydration layer at the interface of water and molecules of DNA, denatured DNA, and protein α- chymotrypsin is found using low-frequency laser spectroscopy of four-photon scattering (FPS). Spectra of rotational resonances of H2O ortho and para spin isomers in aqueous solutions of various biopolymers and in distilled water are obtained in the range of 0 to 100 cm−1 with a spectral resolution of ≈0.12 cm−1. Numerical analysis of the FPS spectra shows that concentration of H2O2 molecules in the DNA hydration layer increases by a factor of three after denaturation. In addition, FPS spectra of aqueous α-chymotrypsin solutions are obtained in the spectral range of ±7 cm−1 at concentrations of 0 to 20mgcm−3. It is found out that the speed of hypersound in the aqueous protein solution measured by the shift of the Brillouin components in the scattering spectrum increases and cubically depends on the concentration.

Effects of aprotic solvents on the stability of metal-free superoxide dismutase probed by native electrospray ionization-ion mobility-mass spectrometry.

Considering that aprotic solvents are often used as cosolvents in investigating the interactions between small molecules and proteins, we assessed the effects of five aprotic solvents represented by dimethylformamide (DMF) on the structure stabilities of metal-free SOD1 (apo-SOD1) by native electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS). These aprotic solvents include DMF, 1,3-dimethyl-2-imidazolidinone (DMI), dimethyl sulfoxide (DMSO), acetonitrile (ACN), and tetrahydrofuran (THF). Results indicated that DMI, DMSO, and DMF at low percentage concentration could reduce the average charge and the dimer dissociation of apo-SOD1. By contrast, ACN and THF at low concentration have no similar effect. DMF was selected as a representative solvent to further investigate the detailed effects on the structure stability of apo-SOD1 by using collision-induced dissociation and unfolding. The results reveal that the addition of minimal DMF to an aqueous protein solution can protect against the unfolding and dissociation of dimer, even under destabilizing conditions (such as low pH or high cone voltage). When the different percentage concentrations of DMF were added, the average collision cross section of apo-SOD1 showed that apo-SOD1 became compacted when the DMF concentration increased from 0% to 1% and eventually started extending when increased from 1% to 20%. The results indicated that DMF has similar effects to DMSO in native mass spectrometry (MS) and it can also be used as a cosolvent besides DMSO in investigating the stabilities of proteins and the interactions between small molecules and proteins.

Binding of safranal to whey proteins in aqueous solution: Combination of headspace solid-phase microextraction/gas chromatography with multi spectroscopic techniques and docking studies

The objective of this work was to study molecular binding of safranal to whey proteins by taking advantage of headspace solid-phase microextraction combined with gas chromatography (HS–SPME/GC), fluorescence and circular dichroism (CD) spectroscopies, and docking studies. The results of HS-SPME/GC indicated that bovine serum albumin (BSA) had the highest affinity toward safranal, with binding constant of 3.196 × 103 M−1. Also, binding strength was reduced in the order of α-lactalbumin (α-Lact), whey protein isolate (WPI), and β-lactoglobulin (β-Lg). Although there was a good agreement between results of HS–SPME/GC and fluorescence spectroscopy regarding the safranal binding site on whey proteins, the order of their binding affinity toward safranal was not consistent for both techniques. According to docking studies, conformational alterations in secondary and tertiary structures of whey proteins induced by safranal association resulted from hydrophobic interactions and hydrogen bonds.

Effects of Winter Flounder Antifreeze Protein on the Growth of Ice Particles in an Ice Slurry Flow in 
 Mini-Channels

The control of ice growth in ice slurry is important for many fields, including (a) the cooling of the brain during cardiac arrest, (b) the storage and transportation of fresh fish and fruits, and (c) the development of distributed air-conditioning systems. One of the promising methods for the control is to use a substance such as antifreeze protein. We have observed and report here growth states of ice particles in both quiescent and flowing aqueous solutions of winter flounder antifreeze proteins in mini-channels with a microscope. We also measured ice growth rates. Our aim was to improve the levels of ice growth inhibition by subjecting the antifreeze protein solution both to preheating and to concentrating by ultrafiltration. We have found that the ice growth inhibition by the antifreeze protein decreased in flowing solutions compared with that in quiescent solutions. In addition, unlike unidirectional freezing experiments, the preheating of the antifreeze protein solution reduced the ice growth inhibition properties. This is because the direction of flow, containing HPLC6 and its aggregates, to the ice particle surfaces can change as the ice particle grows, and thus the probability of interaction between HPLC6 and ice surfaces does not increase. In contrast to this, ultrafiltration after preheating the solution improved the ice growth inhibition. This may be due to the interaction between ice surfaces and many aggregates in the concentrates.

The effects of solution additives and gas-phase modifiers on the molecular environment and conformational space of common heme proteins.

The molecular environment is known to impact the secondary and tertiary structures of biomolecules both in solution and in the gas phase, shifting the equilibrium between different conformational and oligomerization states. However, there is a lack of studies monitoring the impacts of solution additives and gas-phase modifiers on biomolecules characterized using ion mobility techniques. The effect of solution additives and gas-phase modifiers on the molecular environment of two common heme proteins, bovine cytochrome c and equine myoglobin, is investigated as a function of the time after desolvation (e.g., 100-500 ms) using nanoelectrospray ionization coupled to trapped ion mobility spectrometry with detection by time-of-flight mass spectrometry. Organic compounds used as additives/modifiers (methanol, acetonitrile, acetone) were either added to the aqueous protein solution before ionization or added to the ion mobility bath gas by nebulization. Changes in the mobility profiles are observed depending on the starting solution composition (i.e., in aqueous solution at neutral pH or in the presence of organic content: methanol, acetone, or acetonitrile) and the protein. In the presence of gas-phase modifiers (i.e., N2 doped with methanol, acetone, or acetonitrile), a shift in the mobility profiles driven by the gas-modifier mass and size and changes in the relative abundances and number of IMS bands are observed. We attribute the observed changes in the mobility profiles in the presence of gas-phase modifiers to a clustering/declustering mechanism by which organic molecules adsorb to the protein ion surface and lower energetic barriers for interconversion between conformational states, thus redefining the free energy landscape and equilibria between conformers. These structural biology experiments open new avenues for manipulation and interrogation of biomolecules in the gas phase with the potential to emulate a large suite of solution conditions, ultimately including conditions that more accurately reflect a variety of intracellular environments.

Fourier-Transform Infrared (FTIR) Spectroscopy Transmission Technique for Analysis of Characteristic Infrared Bands of Peptide Linkage using Dried Proteins

Protein activity is well dependent on its conformational structure and thus secondary structure prediction and determination of proteins are vital requirements. FTIR is rapidly coming up for protein structural studies, as an alternative to circular dichroism. However, FTIR spectra are primarily recorded using protein samples prepared in water, organic solvents, deuterium oxide, and membranes. FTIR spectra are thus dominated by the solvent spectrum and subtraction of the solvent spectrum introduces severe spectral distortions. In aqueous protein solutions, a strong water signal overlaps amide I bands (C=O stretching; 1700-1600 cm−1), thus masking most of the protein secondary structure information. Further, most of the purified proteins including protein drug formulations are more stable in the solid state than in the liquid state. Hence there is a need to explore the use of FTIR for various physical states of proteins. In the present study, we showed that protein secondary structure can be analyzed in a dried state; using potassium bromide (KBr) pellet method for dried known proteins (bovine serum albumin, proteinase K, fibrinogen etc). KBr pellet method gave excellent spectra for all the used dried proteins; shown by the appearance of characteristic infrared bands of peptide linkage (Amide A, B, I-VI) without much interference from water, buffers, stabilizing factors, co-factors etc. We also found that KBr pellet method can analyze a range of protein types; globular to fibrous. Thus KBr method is a great tool for prediction/determination of the secondary structure of unknown proteins and quality control of solid-state protein drug formulations.

Electrochemical protein recognition based on macromolecular self-assembly of molecularly imprinted polymer: a new strategy to mimic antibody for label-free biosensing.

A versatile strategy, based on the use of an amphiphilic copolymer as a macromonomer, was developed for the preparation of a fully synthetic MIP (molecularly imprinted polymer) sensor for protein recognition. A UV-crosslinkable copolymer poly(DMA-co-HEA-co-St) (UPDHS) was designed and synthesized to assemble with the template protein in aqueous solution, resulting in the fabrication of protein imprinted polymeric nanoparticles. Linear macromolecular chains were used to protect the structural integrity of the protein, through which a 3D structure was formed around the protein molecule to generate recognition cavities. Then the nanoparticles were immobilized on the cleaned surface of a transducer as an MIP sensing platform. The resultant MIP coating was then irradiated via ultraviolet light to ensure that the recognition cavities were stable after UV curing. After protein extraction, recognition cavities complementary to the protein molecule in shape, size and chemical functionality were formed in the platform, which could then selectively rebind to the template in a mixture of closely related compounds. The sensor exhibited satisfactory selectivity, a wide linear range from 10-14 to 10-9 mg mL-1, and a comparatively lower detection limit for protein detection. This strategy offers a new and straightforward method for the synthesis of receptors for label-free and cost-efficient protein recognition. This is one of the most effective and versatile strategies for the preparation of high-performance protein recognition devices based on a fully synthetic MIP.