The present work investigates the capability of single-stranded DNA (ssDNA) in enhancing the intrinsic peroxidase-like activity of the g-C3N4 nanosheets (NSs). We found that ssDNA adsorbed on g-C3N4 NSs could improve the catalytic activity of the nanosheets. The maximum reaction rate of the H2O2-mediated TMB oxidation catalyzed by the ssDNA-NSs hybrid was at least 4 times faster than that obtained with unmodified NSs. The activity enhancement could be attributed to the strong interaction between TMB and ssDNA mediated by electrostatic attraction and aromatic stacking and by both the length and base composition of the ssDNA. The high catalytic activity of the ssDNA-NSs hybrid permitted sensitive colorimetric detection of exosomes if the aptamer against CD63, a surface marker of exosome, was employed in hybrid construction. The sensor recognized the differential expression of CD63 between the exosomes produced by a breast cancer cell line (MCF-7) and a control cell line (MCF-10A). Moreover, a similar trend was detected in the circulating exosomes isolated from the sera samples collected from breast cancer patients and healthy controls. Our work sheds lights on the possibility of using ssDNA to enhance the peroxidase-like activity of nanomaterials and demonstrates the high potential of the ssDNA-NSs hybrid in clinical diagnosis using liquid biopsy.

Protein adsorption alters the “biological identity” of nanoparticles (NPs) and could affect how biosystems respond to invading NPs. Study of protein–NP interaction can help understand how the physicochemical properties of NPs impact the interaction and thus potentially guide the design of safer and more effective NPs for biomedical or other applications. Binding affinity between proteins and NPs and the occurrence of protein conformational change upon binding to NPs are two important aspects to be learned, but few methods are currently available to assess both simultaneously in a simple way. Herein, we demonstrated that the fluorescamine labeling method developed by our group not only could reveal protein conformational change upon adsorption to NPs, owing to its capability to label the primary amines exposed on protein surface, but also could be applied to measure the binding affinity. By screening the interaction between a large number of proteins and four types of NPs, the present study also revealed that protein adsorption onto NPs could be strongly affected by structure flexibility. The proteins with high structure flexibility experienced high degrees of conformation change when binding to the polystyrene NPs, which could potentially influence protein function. Overall, we demonstrate that our assay is a quick, simple, and high-throughput tool to reveal potential impacts on protein activity and evaluate the strength of protein–NP binding.

Arrayed deep cavitands can be coupled to a fluorescence-based supramolecular tandem assay that allows site-selective in situ monitoring of post-translational modifications catalyzed by the lysine methyltransferase PRDM9 or the lysine demethylase JMJD2E. An arrayed sensor system containing only three cavitand components can detect the specific substrates of enzyme modification, in the presence of other histone peptides in the enzyme assay, enabling investigation of cross-reactivity over multiple methylation sites and interference from nonsubstrate peptides.

Variably functionalized self-folding deep cavitands form an arrayed, fluorescent indicator displacement assay system for the detection of post-translationally modified (PTM) histone peptides. The hosts bind trimethyllysine (KMe3) groups, and use secondary upper rim interactions to provide more sensitive discrimination between targets with identical KMe3 binding handles. The sensor array uses multiple different recognition modes to distinguish between miniscule differences in target, such as identical lysine modifications at different sites of histone peptides. In addition, the sensor is affected by global changes in structure, so it is capable of discriminating between identical PTMs, at identical positions on amino acid fragments that vary only in peptide backbone length, and can be applied to detect non-methylation modifications such as acetylation and phosphorylations located multiple residues away from the targeted binding site. The synergistic application of multiple variables allows dual-mode deep cavitands to approach levels of recognition selectivity usually only seen with antibodies.

Cobalt oxyhydroxide (CoOOH) nanoflakes, an emerging type of two-dimensional nanomaterial, show great potential for use in molecular detection. Previous assays utilizing such materials have largely been based on their outstanding fluorescence quenching ability and oxidizing power. Herein, we report the intrinsic peroxidase-like activity of cobalt oxyhydroxide (CoOOH) nanoflakes, and we show how this activity can be employed for glucose detection. We found that, in the presence of hydrogen peroxide (H2O2), the nanoflakes accelerated the conversion of peroxidase substrates such as 3,3′,5,5′-tetramethylbenzidine (TMB) into colored products. By combining the CoOOH nanoflakes with the biological enzyme glucose oxidase (GOx), we developed a colorimetric method for the detection of glucose within the concentration range 5.3–500 μM. The proposed method was applied to detect elevated blood glucose levels in diabetic patients, and the intense color change induced by elevated glucose levels was found to be readily apparent to the naked eye, proving the utility of our assay for point-of-care testing.

A dual-mode aggregative host:guest indicator displacement sensing system has been created for the detection of trimethylated peptides and determination of histone demethylase activity. The combination of selective recognition of suitably sized trimethylammonium salts and reversible lipophilic aggregation of the host:guest complex provides a unique quenching mechanism that is not only dependent on affinity for sensitivity but the lipophilicity of the indicator. In addition, aggregation can be controlled by the application of chaotropic anions in the mixture, allowing a second level of discrimination between hard lysine groups and softer trimethyllysines.

An amine-reactive fluorogenic molecule specifically turned on by superoxide radicals (O2˙−) was synthesized and coupled to a mitochondrial (MT) targeting peptide. The obtained probe showed superior uptake and MT targeting capabilities; and successfully detected the change in O2˙− levels in cells treated with chemical stimuli or single-walled carbon nanotubes.

Phosphorylation is one of the most important post-translational modifications in proteins. Their essential roles in the regulation of cellular processes and alteration of protein–protein interaction networks have been actively studied. However, phosphorylated proteins are present at low abundance in cells, and ionization of the modified peptides is often suppressed by the more abundant species in mass spectrometry. Effective enrichment techniques are needed to remove the unmodified peptides and concentrate the phosphorylated ones before their identification and quantification. Herein, we prepared ZrO2 nanofibers by electrospinning, a straightforward and easy fabrication technique, and applied them to enrich phosphorylated peptides and proteins. The fibers showed good size homogeneity and porosity and could specifically bind to the phosphorylated peptides and proteins, allowing their separation from the unmodified analogues when present in either simple protein digests or highly complex cell lysates. The enrichment performance was superior to that of the commercially available nanoparticles. Moreover, modifying the solution pH could lead to selective adsorption of proteins with different pI values, suggesting the fibers’ potential applicability in charge-based protein fractionation. Our results support that the electrospun ZrO2 nanofibers can serve as a versatile tool for protein analysis with great ease in preparation and handling.Keywords: electrospinning; nanofibers; phosphopeptide enrichment; phosphoprotein; protein fractionation; zirconia

A rapid, high throughput fluorescence assay was designed to screen interactions between proteins and nanoparticles. The assay employs fluorescamine, a primary-amine specific fluorogenic dye, to label proteins. Because fluorescamine could specifically target the surface amines on proteins, a conformational change of the protein upon interaction with nanoparticles will result in a change in fluorescence. In the present study, the assay was applied to test the interactions between a selection of proteins and nanoparticles made of polystyrene, silica, or iron oxide. The particles were also different in their hydrodynamic diameter, synthesis procedure, or surface modification. Significant labeling differences were detected when the same protein incubated with different particles. Principal component analysis (PCA) on the collected fluorescence profiles revealed clear grouping effects of the particles based on their properties. The results prove that fluorescamine labeling is capable of detecting protein–nanoparticle interactions, and the resulting fluorescence profile is sensitive to differences in nanoparticle’s physical properties. The assay can be carried out in a high-throughput manner, and is rapid with low operation cost. Thus, it is well suited for evaluating interactions between a larger number of proteins and nanoparticles. Such assessment can help to improve our understanding on the molecular basis that governs the biological behaviors of nanomaterials. It will also be useful for initial examination of the bioactivity and reproducibility of nanomaterials employed in biomedical fields.

Nanoparticles (NPs) adsorb proteins when in the biological matrix, and the resulted protein corona could affect NP-cell interactions. The corona has a dynamic nature with the adsorbed proteins constantly exchanging with the free proteins in the matrix at various rates. The rapidly exchanging proteins compose the soft corona, which responds more dynamically to environment changes than the hard corona established by the ones with slow exchange rates. In the present study, the corona formed on the superparamagnetic iron oxide NPs (SPIONs) in human serum was studied by flow field-flow fractionation and ultracentrifugation, which rapidly differentiated the corona proteins based on their exchange rates. By varying the surface hydrophobicity of the SPIONs with a core size around 10 nm, we found out that, the more hydrophobic surface ligand attracted proteins with higher surface hydrophobicity and formed a more dynamic corona with a larger portion of the involved proteins with fast exchange rates. Increasing the core diameter of the SPIONs but keeping the surface ligand the same could also result in a more dynamic corona. A brief investigation of the effect on the cellular uptake of SPIONs using one selected corona protein, transferrin, was conducted. The result showed that, only the stably bound transferrin could significantly enhance cellular uptake, while transferrin bound in a dynamic nature had negligible impact. Our study has led to a better understanding of the relationship between the particle properties and the dynamic nature of the corona, which can help with design of nanomaterials with higher biocompatibility and higher efficacy in biosystems for biomedical applications.Keywords: field flow fractionation; protein binding kinetics; protein corona; surface hydrophobicity

Circulating microRNAs (miRNAs) are potential biomarkers useful in cancer diagnosis. They have been found to be bound to various carriers like proteins, lipoprotein particles, and exosomes. It is likely that only miRNAs in particular carriers, but not the overall quantity, are directly related to cancer development. Herein, we developed a method for rapid separation of different miRNA carriers in serum using asymmetrical flow field flow fractionation (AF4). Sera from two healthy individuals (control) or from two cancer patients (case) were fractionated. Six fractions enriching different types of miRNA carriers, such as the lipoprotein particles and exosomes, were collected. The quantities of eight selected miRNAs in each fraction were obtained by RT-qPCR to yield their distribution profiles among the carriers. Larger changes in miRNA quantity between the control and the case were detected in the fractionated results compared to the sum values. Statistical analysis on the distribution profiles also proved that, the quantities of 4 miRNAs within particular fractions showed significant difference between the controls and the cases. On the contrary, if the overall quantity of the miRNA was subject to the same statistical analysis, only 2 miRNAs exhibited significant difference. Moreover, principle component analysis revealed good separation between the controls and the cases with the fractionated miRNA amounts. All in all, we have demonstrated that, our method enables comprehensive screening of the distribution of circulating miRNAs in the carriers. The obtained distribution profile enlarges the miRNA expression difference between healthy individuals and cancer patients, facilitating the discovery of specific miRNA biomarkers for cancer diagnosis.

MicroRNAs (miRNAs) are promising targets for disease diagnosis. However, miRNA detection requires rapid, sensitive, and selective detection to be effective as a diagnostic tool. Herein, a miRNA-initiated exponential strand-displacement amplification (SDA) assay was reported. With the Klenow fragment, nicking enzyme Nt.AlwI, and two primers, the miRNA target can trigger two cycles of nicking, polymerization, and displacement reactions. These reaction cycles amplified the target miRNA exponentially and generated dsDNAs detectable with SYBR Green I in real-time PCR. As low as 16 zmol of the target miRNA was detected by this one-pot assay within 90 min, and the dynamic range spanned over 9 orders of magnitude. Negligible impact from the complex biological matrix was observed on the amplification reaction, indicating the assay’s capability to directly detect miRNAs in biofluids.

•AF4 as a free-solution separation technique can measure the affinity of ssDNA–protein.•Membrane selection should consider membrane pore size and ssDNA gyration radius.•The ssDNA with second structure could remain folded in AF4.•The protein-bound ssDNA can be collected with high purity after AF4.Tools capable of measuring binding affinities as well as amenable to downstream sequencing analysis are needed for study of DNA–protein interaction, particularly in discovery of new DNA sequences with affinity to diverse targets. Asymmetrical flow field-flow fractionation (AF4) is an open-channel separation technique that eliminates interference from column packing to the non-covalently bound complex and could potentially be applied for study of macromolecular interaction. The recovery and elution behaviors of the poly(dA)n strand and aptamers in AF4 were investigated. Good recovery of ssDNAs was achieved by judicious selection of the channel membrane with consideration of the membrane pore diameter and the radius of gyration (Rg) of the ssDNA, which was obtained with the aid of a Molecular Dynamics tool. The Rg values were also used to assess the folding situation of aptamers based on their migration times in AF4. The interactions between two ssDNA aptamers and their respective protein components were investigated. Using AF4, near-baseline resolution between the free and protein-bound aptamer fractions could be obtained. With this information, dissociation constants of ∼16 nM and ∼57 nM were obtained for an IgE aptamer and a streptavidin aptamer, respectively. In addition, free and protein-bound IgE aptamer was extracted from the AF4 eluate and amplified, illustrating the potential of AF4 in screening ssDNAs with high affinity to targets. Our results demonstrate that AF4 is an effective tool holding several advantages over the existing techniques and should be useful for study of diverse macromolecular interaction systems.

Protein adsorption on nanoparticles is closely associated with the physicochemical properties of particles, in particular, their surface properties. We synthesized two batches of polyacrylic acid-coated nanoparticles under almost identical conditions except for the heating duration and found differences in the head-group structure of the polyacrylic acid. The structure change was confirmed by NMR and MS. The two batches of particles had varied binding affinities to a selected group of proteins. Computational work confirmed that the head group of the polymer on the surface of a nanoparticle could directly interact with a protein, and small structural changes in the head group were sufficient to result in a significant difference in the free energy of binding. Our results demonstrate that protein adsorption is so sensitive to the surface properties of particles that it can reveal even small variations in the structure of a nanoparticle surface ligand, and should be useful for quick assessment of nanoparticle properties.

A protein corona will be formed on nanoparticles (NPs) entering a biological matrix, which can influence particles’ subsequent behaviors inside the biological systems. For proteins bound stably to the NPs, they can exhibit different association/dissociation rates. The binding kinetics could affect interaction of the NPs with cell surface receptors and possibly contribute to the outcomes of NPs uptake. In the present study, a method to differentiate the corona proteins based on their relative dissociation rates from the NPs was developed, employing flow field-flow fraction (F4) in combination with centrifugation. The proteins bound to the superparamagnetic iron oxide NPs (SPION) present in an IgG/albumin depleted serum were isolated via collection of the SPIONs by either F4 or centrifugation. They were subsequently analyzed by LC-MS/MS and identified. Because the SPION-protein complexes injected to F4 dissociated continuously under the nonequilibrium separation condition, only the proteins with slow enough dissociation rates would be collected with the NPs in the eluent of F4. However, in centrifugation, proteins with good affinity to the SPIONs were collected regardless of the dissociation rates of the complexes. In both cases, the nonbinding ones were washed off. Capillary electrophoresis and circular dichroism were employed to verify the binding situations of a few SPION-protein interactions, confirming the effectiveness of our method. Our results support that our method can screen for proteins binding to NPs with fast on-and-off rates, which should be the ones quickly exchanging with the free matrix proteins when the NPs are exposed to a new biological media. Thus, our method will be useful for investigation of the temporal profile of protein corona and its evolution in biological matrices as well as for high-throughput analysis of the dynamic feature of protein corona related to particle properties.

A new method to label and detect the long ssDNA products from rolling circle (RC) amplification was reported. ZnS nanocrystal clusters (NCCs) were used to tag the RC products. The NCCs release millions of cations to trigger millions of metal-responsive dyes for cascade signal amplification. Selective and sensitive quantification of miRNA was demonstrated; and the NCCs delivered much lower detection limits compared to using peroxidase or quantum dots as the labels.

•AF4 is useful for study of aptamer–protein binding.•Aptamer binding in AF4 is affected by incubation buffer and carrier solution.•Carrier solution is more critical to maintain binding than incubation buffer.•Using the focusing step for on-line incubation enhances binding efficiency.Asymmetric flow field flow fractionation (AF4) should be suitable for the study of aptamer–target binding, because its gentle separation would impose little disturbance to the complex structure, and it can use carrier solutions with high salt concentrations to provide the most optimal interaction environment to the complex. However, no report has been found for such applications. Herein, we investigated the utility of AF4 as an effective tool for detection of the aptamer–protein complex. With the model system of human immunoglobulin E (IgE) and its aptamer, impacts on aptamer binding from the incubation and AF4 carrier solutions, as well as the flow conditions used during the sample focusing step, were studied. We found that the composition of the carrier solution, in particular, the presence of Mg2+, strongly influenced the complex's integrity in AF4. Also, the focusing conditions during sample injection in AF4 affected the binding equilibrium. Our findings highlight the necessity of maintaining the optimal binding environment during the time course of complex measurement; and demonstrated the good compatibility of AF4 with salty buffers and its high simplicity in conducting on-channel incubation. With its capability to carry out size-based separation of analytes with a wide range of dimensions, AF4 can be employed for detection of large proteins and even biological particles using aptamers. AF4 is also valuable for study of aptamer–target binding under different buffer environments for better understanding of the structure–function relationship of aptamers.

Cation exchange (CX) in the nonfluorescent ZnS nanocrystal clusters (NCCs) was employed to detect trace biomolecules with immunoassays. The NCCs were porous and allowed fast cation exchange reaction to release an ultralarge number of Zn2+ from each cluster that turned on the Zn-responsive dyes for fluorescence detection. The ZnS NCCs were highly stable in biological buffers and more biocompatible than quantum dots. Zn2+ release efficiency and target binding by NCCs with average diameters of 44 nm, 86 nm, and 144 nm were investigated. The smallest NCCs exhibited the highest CX efficiency because of its larger surface area and bigger pores inside the cluster structure, and 71.0% of the enclosed Zn2+ were freed by CX with 2-min microwave irradiation. They also experienced the least space hindrance and the fastest rate when binding to target molecules immobilized on surface. When the 44-nm NCCs were used to detect IgE in a sandwich assay, the limit of detection (LOD) was 5 pg/mL (33 fM), 1,000 times better than that of ELISA. Our results well demonstrate that CX in the ZnS NCCs is superior to the conventional signaling strategies in its high amplification efficiency, robustness, and biocompatibility.

Better understanding of electron transfer (ET) taking place at the nano-bio interface can guide design of more effective functional materials used in fuel cells, biosensors, and medical devices. Single-walled carbon nanotube (SWCNT) coupled with biological enzymes serves as a model system for studying the ET mechanism, as demonstrated in the present study. SWCNT enhanced the activity of horseradish peroxidase (HRP) in the solution-based redox reaction by binding to HRP at a site proximate to the enzyme’s activity center and participating in the ET process. ET to and from SWCNT was clearly observable using near-infrared spectroscopy. The capability of SWCNT in receiving electrons and the direct attachment of HRP to the surface of SWCNT strongly affected the enzyme activity, further confirming the involvement of SWCNT in ET.

Flow field flow fractionation (F4) is an invaluable separation tool for large analytes, including nanoparticles and biomolecule complexes. However, sample loss due to analyte-channel membrane interaction limits extensive usage of F4 at present, which could be strongly affected by the carrier fluid composition. This work studied the impacts of carrier fluid (CF) composition on nanoparticle (NP) recovery in F4, with focus on high ionic strength conditions. Successful analysis of NPs in a biomolecules-friendly environment could expand the applicability of F4 to the developing field of nanobiotechnology. Recovery of the unfunctionalized polystyrene NPs of 199, 102, and 45 nm in CFs with various pH (6.2, 7.4 and 8.2), increasing ionic strength (0–0.1 M), and different types of co- and counter-ions, were investigated. Additionally, elution of the 85 nm carboxylate NPs and two proteins, human serum albumin (HSA) and immunoglobulin (IgG), at high ionic strengths (0–0.15 M) was investigated. Our results suggested that (1) electrostatic repulsion between the negatively charged NPs and the regenerated cellulose membrane was the main force to avoid particle adsorption on the membrane; (2) larger particles experienced higher attractive force and thus were influenced more by variation in CF composition; and (3) buffers containing weak anions or NPs with weak anion as the surface functional groups provided higher tolerance to the increase in ionic strength, owing to more anions being trapped inside the NP porous structure. Protein adsorption onto the membrane was also briefly investigated in salted CFs, using HSA and IgG. We believe our findings could help to identify the basic carrier fluid composition for higher sample recovery in F4 analysis of nanoparticles in a protein-friendly environment, which will be useful for applying F4 in bioassays and in nanotoxicology studies.Highlights► High repulsive force between NPs and membrane can minimize particle adsorption. ► Weaker anion in solution or on NP surface causes lower NP adsorption at high conc. ► Highly charged counter ions increases NP adsorption on membrane significantly. ► Protein conformation plays an important role in protein–membrane interaction.

The study of the regulatory roles in small RNAs can be accelerated by techniques that permit simple, low-cost, and rapid extraction of small RNAs from a small number of cells. In order to ensure highly specific and sensitive detection, the extracted RNAs should be free of the background nucleic acids and present stably in a small volume. To meet these criteria, we designed a multi-well/multi-channel (M&M) chip to carry out automatic and selective isolation of small RNAs via solid-phase extraction (SPE), followed by reverse-transcription (RT) to convert them to the more stable cDNAs in a final volume of 2 μL. Droplets containing buffers for RNA binding, washing, and elution were trapped in microwells, which were connected by one channel, and suspended in mineral oil. The silica magnetic particles (SMPs) for SPE were moved along the channel from well to well, i.e. in between droplets, by a fixed magnet and a translation stage, allowing the nucleic acid fragments to bind to the SMPs, be washed, and then be eluted for RT reaction within 15 minutes. RNAs shorter than 63 nt were selectively enriched from cell lysates, with recovery comparable to that of a commercial kit. Physical separation of the droplets on our M&M chip allowed the usage of multiple channels for parallel processing of multiple samples. It also permitted smooth integration with on-chip RT-PCR, which simultaneously detected the target microRNA, mir-191, expressed in fewer than 10 cancer cells. Our results have demonstrated that the M&M chip device is a valuable and cost-saving platform for studying small RNA expression patterns in a limited number of cells with reasonable sample throughput.

ZnSe nanocrystals (NCs), possessing low native luminescence but high biocompatibility, were employed as labeling tags in bioassays. They were able to amplify each target recognition event thousands of times through a cation-exchange reaction (CXAmp) that released over 3000 encapsulated Zn2+ from one single NC. The freed cations in turn triggered strong fluorescence from the Zn-responsive dyes. The present study demonstrated that CXAmp with ZnSe delivered superior detection performance in comparison to the conventional labeling methods. The overall fluorescence intensity of CXAmp using 5 nM ZnSe NCs was 30 times higher than that from 5 nM core−shell CdSe/ZnS quantum dots (QDs). The limit of detection (LOD) obtained with ZnSe-based CXAmp was 10-fold lower than with horseradish peroxidase (HRP) labeling, and the detection sensitivity, represented by the slope of the signal-versus-concentration curve, was 20-fold higher. When applied to detect immunoglobulin E (IgE) in a sandwich format, a LOD of 1 ng/mL was achieved. The highly sensitive CXAmp also allowed detection of the total IgE content in dilute human serum, in which the abundant matrix proteins exhibited less interference and more accurate quantification could be performed. Besides high signal amplification efficiency and good biocompatibility, CXAmp with ZnSe could be easily adapted to common laboratory settings and act as a universal labeling system for reliable detection of low-abundance targets.

For the first time, the possible binding site of nanoparticles on protein was revealed by cross-linking chemistry coupled with mass spectrometry. The peptides located very close to the poly(acrylic acid) (PAA)-coated Fe3O4 nanoparticles (NPs) during interaction with human serum albumin (HSA) were cross-linked to the surface of NPs. Following protease digestion, the attached peptides were cleaved off the particle surface and identified by matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). The peptides were found to be part of the so-called drug binding site 2 of HSA; and the competitive binding to HSA between the corresponding drug, ibuprofen, and the NPs was observed. Our results demonstrated that cross-linking chemistry coupled with MS was a quick and simple method for locating the possible binding sites of NPs on protein. Information on NP-protein binding interface will benefit the study of how the interactions are governed by the physicochemical properties of NPs, for guiding the design of functional bionano constructs. It can also help to predict the biological consequence of protein adsorption on NPs, for obtaining more knowledge on nanotoxicity.

A sensitive and simple assay for the detection of Pb2+ in aqueous solutions is reported. It takes advantage of the high affinity between single-stranded DNA (ssDNA) and single-walled carbon nanotubes (SWCNT) as well as the capability of SWCNT in fluorescence quenching. Lead(II) catalyzes the cleavage of a fluorescently labeled DNA substrate by a DNAzyme, which releases the single-stranded product to be adsorbed onto a SWCNT. The decrease in fluorescence is proportional to the Pb2+ concentration. Concentrations as low as 1 nM Pb2+ in water could be detected and the detection range spans over 5 orders of magnitude. The unique combination of Pb-specific DNAzyme with SWCNT produces a universal, facile and cost-effective sensing platform for lead ions. The concept can be applied to the design of detection assays for other metal ions or small molecules.

Understanding nanoparticle−protein interaction could help in promoting applications of nanoparticles in the biomedical fields and reducing/preventing possible adverse effects to the biological systems caused by nanoparticles. Quantitative measurement of the biophysical parameters of nanoparticle−protein interaction will improve such understanding, which could be conveniently performed by capillary electrophoresis (CE) as demonstrated in the present study. Two interaction situations were identified. Stable nanoparticle−protein complexes were resolved from the free nanoparticles and the proteins by capillary zone electrophoresis (CZE). Transient complexes with fast association/dissociation rates showed distinct mobility change from the free nanoparticles in affinity capillary electrophoresis (ACE). Interactions of bovine serum albumin (BSA) with the Fe3O4 nanoparticles (average diameters of 8 and 10 nm) and with the Au nanoparticles (average diameters of 5 and 10 nm) displayed slow and fast binding kinetics, respectively. Using the Hill equation, we could calculate the dissociation constants (KD) and cooperativity coefficients (n). Impacts on nanoparticle−protein interaction from the physicochemical properties of nanoparticles and the incubation buffer were evaluated on the basis of the KD and n values to interpret the interaction driving forces. Our study demonstrated the high simplicity and flexibility of CE in probing the interaction of proteins with diverse particles. The separation power of CE should also facilitate studies of the multicomponent interaction systems for investigating how adsorption onto nanoparticles could affect the protein−protein or protein−small molecule interactions.

How single-walled carbon nanotubes (SWCNT) function in redox reactions may be related to their behaviors in the induction of oxidative stress. Herein, oxidation of several biologically relevant reducing agents in the presence of SWCNT was studied in aqueous solutions. The selected reductants included a common indicator for intracellular reactive oxygen species (ROS) (2,7-dichlorodihydrofluorescein), small antioxidants (vitamin C, Trolox, and cysteine), and a high-molecular-weight ROS scavenger (bovine serum albumin). The unmodified or carboxylated SWCNT acted as both the oxidants and the catalysts in reactions. Moreover, they accelerated the oxidation reactions mediated by horseradish peroxidase, a representative member of the enzyme family actively involved in balancing oxidative stress. These diverse roles in redox reactions may serve the chemistry basis for SWCNT to induce oxidative stress in biological systems as potential environmental pollutants.

A simple and sensitive approach to detect Hg2+ colorimetrically using blotting membrane is described to detect as low as 0.1 nM Hg2+ with the aid of a scanner.

Small RNA molecules are effective regulators of gene expression, and the expression signature of one subgroup of small RNA, the microRNA (miRNA), has been linked to disease development and progression. Therefore, detection of small RNA in biological samples will greatly improve the understanding of their functions and render effective tools to researchers for cellular process control and disease prevention. To solve the challenges in detecting the low-abundance and short strand-length of small RNA molecules, we designed a ligation-assisted binding assay and applied the cation exchange-based fluorescence amplification (CXFluoAmp) method developed in our group for detection. Nonfluorescent, ionic nanocrystals (NCs) of CdSe were conjugated to detection probes and immobilized onto the array surface via ligation with the target small RNA, miR21, which bound to the capture probe complimentarily. Each binding event induced by one target miR21 molecule was then amplified by the release of thousands of Cd2+ from one NC. The free Cd2+ immediately turned on the fluorescence of thousands of fluorogenic Rhod-5N molecules. With such a powerful signal amplification strategy, our assay achieved a limit of detection (LOD) of 35 fM and signals were detectible with analyte concentrations spanning over 7 orders of magnitude. We also identified the differential expression of miR21 in total RNA extracts from healthy breast tissue and diseased cells. Furthermore, our detection scheme demonstrated good specificity in small RNA detection, because significant signal intensity could be observed from small RNAs with one or two nucleotides difference in sequences. Thus, our assay has great application potential for disease diagnosis relying on miRNA biomarkers, or in small RNA expression profiling for new target discovery and functional study.

The mechanism of how single-walled carbon nanotubes (SWCNTs) induce oxidative stress in cells has always been under debate. In the present paper, we have clearly identified the generation of peroxyl radical (ROO•) by the unmodified SWCNT and the poly(ethylene glycol)-functionalized SWCNT (SWCNT-PEG) in aqueous solutions using a simple analytical technique: capillary electrophoresis (CE). CE provides a sensitive detection platform for the oxidation product of the employed reactive oxygen species (ROS) indicator, 2,7-dichlorodihydrofluorescein (H2DCF). In addition, its high separation power eliminates the fluorescence quenching exerted by SWCNTs, which was a big problem that has been encountered in measurements with microplate readers, and facilitates the screening of a large variety of ROS scavengers, including small molecules, surfactant, and proteins, in our study. Furthermore, CE enables the simultaneous incubation of multiple ROS indicators for comparison of their oxidation efficiency, improving the detection specificity. The adsorption of oxygen on the SWCNT surface could be the source of the identified ROO•, because surface coverage by surfactant or proteins could inhibit the oxidation and the anaerobic experiment led to data agreeing with the Langmuir isotherm. The peroxyl radical can serve as the seed for the production of more-active ROS after the SWCNT enters the cell and, therefore, SWCNTs hold potential toxicity to the biological hosts.

Noncoding small RNAs play diverse, important biological roles through gene expression regulation. However, their low expression levels make it difficult to identify new small RNA species and study their functions, calling for the development of detection schemes with higher simplicity, sensitivity, and specificity. Herein, we reported a straightforward assay that combined the stand-alone rolling circle amplification (RCA) with capillary electrophoresis (CE) for specific and sensitive detection of small RNAs in biological samples. In order to enhance the overall reaction efficiency and simplify the procedure, RCA was not preceded with ligation, and a preformed circular probe was employed as the template for the target small RNA-primed isothermal amplification. The long RCA product was digested and analyzed by CE. Two DNA polymerases, the Phi29 and Bst, were compared for their detection performance. Bst is superior in the aspects of specificity, procedure simplicity, and reproducibility, while Phi29 leads to a 5-fold lower detection limit and is able to detect as low as 35 amol of the target small RNA. Coamplification of an internal standard with the target and employment of the RNase A digestion step allow accurate and reproducible quantification of low amounts of small RNA targets spiked into hundreds of nanograms of the plant total RNA extract with a recovery below 110% using either enzyme. Our assay can be adapted to a capillary array system for high-throughput screening of small RNA expression in biological samples. Also, the one-step isothermal process has the potential to conveniently amplify a very limited amount of the RNA samples, e.g., RNA extracted from only a few cells, inside the capillary column or on a microchip.

Fluorescence-based detection is the most common method utilized in biosensing because of its high sensitivity, simplicity, and diversity. In the era of nanotechnology, nanomaterials are starting to replace traditional organic dyes as detection labels because they offer superior optical properties, such as brighter fluorescence, wider selections of excitation and emission wavelengths, higher photostability, etc. Their size- or shape-controllable optical characteristics also facilitate the selection of diverse probes for higher assay throughput. Furthermore, the nanostructure can provide a solid support for sensing assays with multiple probe molecules attached to each nanostructure, simplifying assay design and increasing the labeling ratio for higher sensitivity. The current review summarizes the applications of nanomaterials—including quantum dots, metal nanoparticles, and silica nanoparticles—in biosensing using detection techniques such as fluorescence, fluorescence resonance energy transfer (FRET), fluorescence lifetime measurement, and multiphoton microscopy. The advantages nanomaterials bring to the field of biosensing are discussed. The review also points out the importance of analytical separations in the preparation of nanomaterials with fine optical and physical properties for biosensing. In conclusion, nanotechnology provides a great opportunity to analytical chemists to develop better sensing strategies, but also relies on modern analytical techniques to pave its way to practical applications.

Here we proved the principle of a multiplexed affinity-based protein complex purification (MAPcP) technique that targets simultaneous extraction of multiple protein complexes with superior purity. Microspheres of various sizes and coupled with different affinity probes extract several protein complexes concurrently and specifically. After the coextraction, flow-field flow fractionation (Fl-FFF) rapidly washes the microspheres as well as separates them based on their sizes to recover the clean individual complex for downstream analysis. Demonstration of the parallel extraction of two immuno-complexes from the yeast whole cell lysate showed that MAPcP can enhance the sample purity significantly compared to the traditional centrifugation and magnetic pull-down methods used for small scale protein purification. Simultaneous isolation of multiple protein complexes can facilitate the elucidation of the functional relationship among protein complexes and improve our understanding of the biological network.

An amine-reactive fluorogenic molecule specifically turned on by superoxide radicals (O2˙−) was synthesized and coupled to a mitochondrial (MT) targeting peptide. The obtained probe showed superior uptake and MT targeting capabilities; and successfully detected the change in O2˙− levels in cells treated with chemical stimuli or single-walled carbon nanotubes.

A simple and sensitive approach to detect Hg2+ colorimetrically using blotting membrane is described to detect as low as 0.1 nM Hg2+ with the aid of a scanner.