•cIEF was applied for PNA pI determination for the first time.•MEKC mode for direct PNA characterization was established for the first time.•MEKC mode was applied for fast and direct PNA-protein complex identification.Peptide Nucleic Acid (PNA) is a nucleic acid analogue, whose neutrally charged and hydrophobic backbone makes it more stable in vivo, so it might act as a potentially better protein probe as compared to aptamer. Currently the investigation of PNA and protein interaction is scarce. In this research, multiple modes of capillary electrophoresis were established and applied for PNA characterization and its interaction with ssDNA and protein. A 15-mer PNA having the same nucleobase sequence as 15-mer anti-thrombin DNA aptamer was chosen as PNA model for this study, its pI (7.71) was estimated by capillary isoelectric focusing (cIEF). Due to its neutral charge and strong hydrophobicity, three micellar electrokinetic chromatography (MEKC) modes containing (a) SDS, (b) Triton X-100 and (c) CTAB were compared for PNA related analysis. CTAB was not applicable for PNA analysis, while in 4 mM SDS or 2 mM Triton X-100, PNA and PNA-ssDNA complex can be identified directly. The significant peak of PNA-ssDNA complex helped in validating the two MEKC modes for PNA and target interaction study. Furthermore, the effect of SDS and Triton X-100 concentrations in the two MEKC modes on the protein target thrombin analysis was investigated by capillary zone electrophoresis (CZE). 4 mM SDS caused thrombin denaturation. So in 2 mM Triton X-100, interactions of PNA with thrombin, PNA with RNase A and a non-aptameric PNA (n-PNA) with thrombin were compared. PNA with thrombin exhibited strongest binding. In summary, cIEF mode for PNA pI determination, MECK mode for direct PNA, PNA-ssDNA and PNA-protein complex identification and CZE mode for the effect of surfactant in MEKC modes on protein target thrombin analysis were applied. Above results showed that multiple modes of CE provide rapid and very low-sample cost methods for PNA related studies.

•CE applied to characterize complete ssDNA sequence distribution in whole-cell SELEX process.•CE applied for whole-cell target and ssDNA interaction evaluation.•Binding comparison of three cell lines with a ssDNA sequence and a ssDNA libraryWhole-cell SELEX faces more difficulties than SELEX against purified molecules target. In this work, we demonstrate the application of capillary electrophoresis for assisting whole-cell aptamers selection by characterizing complete ssDNA distribution. We chose three cancer cell lines U251, Hela and PC3 as target, FAM labeled Sgc8c (a 41mer aptamer) and FAM labeled 41mer random ssDNA library as ssDNA model. CE conditions of running buffer and capillary length and inner diameter as well as UV and LIF detection were optimized. The distribution percentage of Sgc8c and ssDNA library against U251, Hela and PC3 was demonstrated, the relative peak area of their complex is 8.94%, 1.05% and 0.44% for Sgc8c and 9.03%, 1.04% and 0.12% for ssDNA library respectively. Under the chosen experimental conditions, binding ability comparison of three cell lines was U251 > Hela > PC3, which was validated by laser confocol microscope. For each cell, distribution percentage of ssDNA library was compared with that of Sgc8c. Finally, whole-cell complex of U251–Sgc8c was confirmed by increase incubation time and fraction CE analysis.

•Interaction affinity comparison of ssDNA and microbial cells with different surface situation.•Microbial cells surface situation determines binding affinity with ssDNA.•CZE and ACE are preferable techniques for interaction evaluation.For whole-cell aptamers selection, cells surface situation has great impact on single-stranded (ssDNA) binding and aptamers selection. In this work, both Lactobacillus acidophilus and Escherichia coli as well as their protoplasts were as cells targets, their interaction with ssDNA library were evaluated based on capillary zone electrophoresis (CZE) and affinity capillary electrophoresis (ACE) with UV and LIF detection. Our results demonstrated that protoplasts without cells wall had apparently stronger interaction with ssDNA library than bacteria, the protoplasts-ssDNA complex could be observed clearly with CZE-LIF. Furthermore, E. coli pretreated by four organic solvents (methanol, ethanol, formaldehyde and glutaraldehyde) showed binding difference with ssDNA library, which could be identified with ACE-UV. Binding constants indicated the interaction of E. coli with ssDNA library were in the order of E. coli protoplasts > methanol (ethanol) treated E. coli > formaldehyde (glutaraldehyde) treated E. coli ≈ E. coli. Above results suggest that cells surface situation determines their binding affinity with ssDNA, which should be considered in whole-cell aptamers selection and aptamers further application. Capillary electrophoresis is a preferable technique for interaction evaluation of composite targets binding with ssDNA library.

•A novel low pH CE-SELEX is proposed for interaction validation of proteins and ssDNA library.•Reverse migration of complex and unbound ssDNA makes complete protein–ssDNA complex collection without ssDNA interference.•Direct complex collection at cathode is convenient no need to exactly control collection time.•Larger amount protein–ssDNA complex collection than common CE-SELEX.In this work, a novel low pH CE-SELEX (LpH-CE-SELEX) as a CE-SELEX variant is proposed. Transferring (Trf), bovine serum albumin (BSA) and cytochrome c (Cyt c) as model protein are incubated with a FAM labeled ssDNA library, respectively. Incubation mixture is separated in low pH CE (pH 2.6), where positively charged protein, protein–ssDNA complex and negatively charged ssDNA library migrate oppositely without EOF driven. Analysis of protein–ssDNA complex under positive voltage and unbound ssDNA library under negative voltage by CE–UV are applied for interactive evaluation. By increasing injection time, larger amount protein–ssDNA complex can be collected conveniently at the cathode end whereas ssDNA migrates to anode. Finally, stability of protein–ssDNA complex in low pH CE separation is discussed.

Capillary electrophoresis-laser inductive fluorescence (CE-LIF) was used to characterize the active effect difference of coupling agent N-hydroxysulfo-succinimide (NHS) and the mixture of NHS and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimides hydrochloride (EDC) on quantum dots CdTe, and to optimize the conditions of CdTe and transferring (Trf) coupling. The labeling effect of CdTe-Trf and CdTe-BSA complex as well as bare CdTe in Hela cell was compared. The results demonstrated that the property of CdTe activated by NHS was better than that of the mixture of EDC-NHS. When 12.5, 31.2 and 40.6 μM Trf were used to couple CdTe respectively, the resultant of CdTe with 31.2 μM Trf optimized by CE-LIF showed the best labeling on Hela. The CdTe-Trf resultant labeled Hela rapidly on the surface when they incubated at 4 °C for 20 min, and transferred into Hela when they incubated at 37 °C for 7 h, which manifested the CdTe-Trf resultant has biological function. The CdTe-Trf can recognize and bind the Trf receptor on cell membrane specifically and enter Hela, while CdTe-BSA cannot recognize Hela specifically and bare CdTe cannot label on cell.

Currently, small proteins imprinting are more reported since large proteins molecular imprinting faces challenge due to their bulk size and complex structure. In this work, bovine serum albumin (BSA) surface-imprinted magnetic polymer was successfully synthesized based on atomic transfer radical polymerization (ATRP) method in the presence of common monomer (N-isopropylacrylamide) with the assistant of basic functional monomer (N-[3-(dimethylamino)propyl]-methacrylamide), which provides a achievable attempt for imprinting larger target proteins based on the ATPR with the mild reaction conditions. The BSA-imprinted polymer exhibited higher adsorption capacity and selectivity to BSA over the non-imprinted polymer. Competitive adsorption tests indicated the BSA-imprinted polymer had better selective adsorption and recognition properties to BSA in the mixture. The obtained BSA-imprinted polymer was applied to bovine serum, which also showed selectivity to BSA. In addition, a conventional aqueous two-phase solution of PEG/sulphate was used as elution for adsorbed BSA, which was compared with common NaCl elution.

Both of the magnetic particle adsorption and aqueous two-phase extraction (ATPE) were simple, fast and low-cost method for protein separation. Selective proteins adsorption by carboxyl modified magnetic particles was investigated according to protein isoelectric point, solution pH and ionic strength. Aqueous two-phase system of PEG/sulphate exhibited selective separation and extraction for proteins before and after magnetic adsorption. The two combination ways, magnetic adsorption followed by ATPE and ATPE followed by magnetic adsorption, for the separation of proteins mixture of lysozyme, bovine serum albumin, trypsin, cytochrome C and myloglobin were discussed and compared. The way of magnetic adsorption followed by ATPE was also applied to human serum separation.

Molecular imprinting as a promising and facile separation technique has received much attention because of their high selectivity for target molecules. In this study, the superparamagnetic lysozyme surface-imprinted polymer was prepared by a novel fabricating protocol, the grafting of the imprinted polymer on magnetic particles in aqueous media was done by atom transfer radical polymerization (ATRP), and the properties of the imprinted polymer were characterized in detail. Its high selective adsorption and recognition to lysozyme demonstrated the separation ability of the magnetic imprinted material to template molecule, and it has been used for quick and direct separation of lysozyme from the mixture of standard proteins and real egg white samples under an external magnetic field. Furthermore, the elution of lysozyme from the imprinted material was achieved by PEG/sulphate aqueous two-phase system, which caused lysozyme not only desorption from the imprinted materials but also redistribution in the top and bottom phase of aqueous two-phase system. The aqueous two-phase system exhibited some of the extraction and enrichment effect to desorbed lysozyme. Our results showed that ATRP is a promising method for the protein molecularly imprinted polymer preparation.

A simple aqueous two-phase extraction system (ATPS) of PEG/phosphate was proposed for selective separation and enrichment of proteins. The combination of ATPE with HPLC was applied to identify the partition of proteins in two phases. Five proteins (bovine serum albumin, Cytochrome C, lysozyme, myoglobin, and trypsin) were used as model proteins to study the effect of phosphate concentration and pH on proteins partition. The PEG/phosphate system was firstly applied to real human saliva and plasma samples, some proteins showed obviously different partition in two phases. The primary results manifest the selective separation and enrichment of proteins in ATPS provided the potential for high abundance proteins depletion in proteomics.

Both of the magnetic particle adsorption and aqueous two-phase extraction (ATPE) were simple, fast and low-cost method for protein separation. Selective proteins adsorption by carboxyl modified magnetic particles was investigated according to protein isoelectric point, solution pH and ionic strength. Aqueous two-phase system of PEG/sulphate exhibited selective separation and extraction for proteins before and after magnetic adsorption. The two combination ways, magnetic adsorption followed by ATPE and ATPE followed by magnetic adsorption, for the separation of proteins mixture of lysozyme, bovine serum albumin, trypsin, cytochrome C and myloglobin were discussed and compared. The way of magnetic adsorption followed by ATPE was also applied to human serum separation.