Wenwan Zhong

Nanomaterials in fluorescence-based biosensing

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.

Self-assembled TiO2 nanocrystal clusters for selective enrichment of intact phosphorylated proteins.

Mesoporous oxides have been used for selective separation and adsorption of biomolecules. Inorganic oxide materials such as silica can be synthesized into highly ordered mesoporous structures featuring high in-pore surface areas, narrow pore size distributions, adjustable pore sizes, and modifiable surface properties. 12–15] Such unique properties favor the selective enrichment of proteins or peptides based on the size-exclusion mechanism. Classic mesoporous silica structures with narrow pore size distributions, such as MCM41 and SBA-15, have been used to selectively enrich peptides from human plasma and exclude other proteins according to an accurate molecular weight (MW) cutoff. However, this type of enrichment method is limited mainly to silica and alumina because currently it is still difficult to extend broadly the surfactant-templating method to the synthesis of mesoporous structures of many other oxides in a convenient, controllable, and scalable manner (although the literature does contain some relevant procedures). The limited choice of materials makes it difficult to further enhance the selectivity by taking advantage of both the size-exclusion effect and the specific binding between many oxides and proteins or peptides. Herein, we propose a general strategy for the fabrication of novel porous nanostructured materials for the efficient separation of biomolecules such as proteins, peptides, and DNA. Briefly, nanoparticles of various nanostructured materials with uniform sizes and shapes are firstly synthesized and then self-assembled into three-dimensional submicrometer clusters containing uniform mesoscale pores. Thanks to rapid progress in colloidal nanostructure synthesis, a great number of materials can now be routinely produced in the form of nanoparticles with excellent control over size, shape, and surface properties, making it possible to utilize specific material–protein/peptide interactions to enhance the selectivity in protein/peptide enrichment. 24] Additionally, size exclusion can also be achieved by controlling the dimensions of the pores produced by the packing of constituent nanoparticles, allowing selective enrichment of biomolecules based on their sizes. The outer surface of each cluster can be made highly hydrophilic, so that nonspecific binding of many hydrophobic proteins/peptides can be avoided. The selfassembly process also brings the convenience of incorporation of multiple components into the clusters to further facilitate separation and detection. For example, the incorporation of fluorescent nanocrystals, such as quantum dots, during the assembly process may produce multifunctional microspheres that are able not only to selectively enrich but also to easily identify the targeted biomolecules. Also, adding superparamagnetic iron oxide nanocrystals to the clusters allows their efficient removal from the analyte solution after selective adsorption by using an external magnetic field. As an example, we report herein the fabrication of mesoporous TiO2 nanocrystal clusters and demonstrate their use for selective enrichment of intact phosphorylated proteins from complex biological samples by taking advantage of both the specific affinity offered by the metal oxide and the sizeexclusion mechanism enabled by the mesoporous structure. The efficient separation and accurate analysis of phosphorylated proteins are highly demanded in biomedical applications because phosphorylation of proteins is a key event in most cellular processes, including signal transduction, gene expression, cell cycle, cytoskeletal regulation, and apoptosis. 28] The combination of peptide mapping by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) with the methods of phosphopeptide enrichment represents an efficient tool for the characterization of the phosphorylated proteins after digestion. The most widely used approaches for specific enrichment of phosphopeptides are immobilized metal affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC). 29–32] In IMAC, phosphopeptides can be selectively retained because of the affinity of metal ions for the phosphate groups, whereas in MOAC, the specific adsorption results from bridging bidentate bindings formed between the phosphate anions and the surface of a metal oxide, such as TiO2, ZrO2, Fe2O3, and Al2O3. [33] However, these techniques can provide information about the original phosphorylated proteins only indirectly, and direct enrichment of phosphorylated proteins by IMAC or MOAC is rarely reported because of the low adsorption efficiency and significant losses of phosphorylated proteins during the washing steps. 35] Therefore, despite the intense interest in studying phosphorylation events, the direct enrichment of intact phosphorylated proteins remains a technical challenge. In this Communication, we describe the selective enrichment of phosphorylated proteins from protein mixtures by using mesoporous colloids of TiO2 fabricated through self-assembly [*] Z. Lu, M. Ye, N. Li, Prof. W. Zhong, Prof. Y. Yin Department of Chemistry, University of California Riverside, CA 92521 (USA) Fax: (+ 1)951-827-4713 E-mail: yadong.yin@ucr.edu Homepage: http://faculty.ucr.edu/~ yadongy/

Detection of microRNA by fluorescence amplification based on cation-exchange in nanocrystals.

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 detectable 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.

Exponential strand-displacement amplification for detection of micrornas

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 assays capability to directly detect miRNAs in biofluids.

Nano Aptasensor for Protective Antigen Toxin of Anthrax

We demonstrate a highly sensitive nano aptasensor for anthrax toxin through the detection of its polypeptide entity, protective antigen (PA toxin) using a PA toxin ssDNA aptamer functionalized single-walled carbon nanotubes (SWNTs) device. The aptamer was developed in-house by capillary electrophoresis systematic evolution of ligands by exponential enrichment (CE-SELEX) and had a dissociation constant (K(d)) of 112 nM. The aptasensor displayed a wide dynamic range spanning up to 800 nM with a detection limit of 1 nM. The sensitivity was 0.11 per nM, and it was reusable six times. The aptasensor was also highly selective for PA toxin with no interference from human and bovine serum albumin, demonstrating it as a potential tool for rapid and point-of-care diagnosis for anthrax.

Probing Nanoparticle−Protein Interaction by Capillary Electrophoresis

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 Fe(3)O(4) 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 (K(D)) 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 K(D) 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.

Stand-alone rolling circle amplification combined with capillary electrophoresis for specific detection of small RNA.

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 Signal Amplification by Cation Exchange in Ionic Nanocrystals

Lighting-up time: A cation-exchange reaction releases thousands of divalent cations from nonfluorescent CdSe ionic crystals and triggers fluorescence from thousands of originally nonfluorescent Fluo-4 fluorophores to obtain a large fluorescence amplification (see picture) and a low detection limit in bioassays. The technique is fast, simple, with a large dye-to-reporter labeling ratio, and flexible in selection of nanocrystals and fluorophores.

Combinatorial Enantiomeric Separation of Diverse Compounds Using Capillary Array Electrophoresis

Combinatorial chiral separations were performed on a 96‐capillary array electrophoresis system. A comprehensive enantioseparation protocol employing neutral and sulfated cyclodextrins as chiral selectors for common basic, neutral and acidic compounds was developed. By using only four judiciously chosen separation buffers, successful enantioseparations were achieved for 49 out of 54 test compounds spanning a large variety of pK and structures. Therefore, unknown compounds can be screened in this manner to identify the optimal enantioselective conditions in just one run.

Cation Exchange in ZnSe Nanocrystals for Signal Amplification in Bioassays

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 Zn(2+) 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.