Micromechanical detection of proteins using aptamer-based receptor molecules

We report label-free protein detection using a microfabricated cantilever-based sensor that is functionalized with DNA aptamers to act as receptor molecules. The sensor utilizes two adjacent cantilevers that constitute a sensor/reference pair and allows direct detection of the differential bending between the two cantilevers. One cantilever is functionalized with aptamers selected for Taq DNA polymerase while the other is blocked with single-stranded DNA. We have found that the polymerase-aptamer binding induces a change in surface stress, which causes a differential cantilever bending that ranges from 3 to 32 nm depending on the ligand concentration. Protein recognition on the sensor surface is specific and has a concentration dependence that is similar to that in solution.     

Surface-immobilized peptide aptamers as probe molecules for protein detection

We demonstrate the use of surface-immobilized, oriented peptide aptamers for the detection of specific target proteins from complex biological solutions. These peptide aptamers are target-specific peptides expressed within a protein scaffold engineered from the human protease inhibitor stefin A. The scaffold provides stability to the inserted peptides and increases their binding affinity owing to the resulting three-dimensional constraints. A unique cysteine residue was introduced into the protein scaffold to allow orientation-specific surface immobilization of the peptide aptamer and to ensure exposure of the binding site to the target solution. Using dual-polarization interferometry, we demonstrate a strong relationship between binding affinity and aptamer orientation and determine the affinity constant KD for the interaction between an oriented peptide aptamer ST(cys+)_(pep9) and the target protein CDK2. Further, we demonstrate the high selectivity of the peptide aptamer STM_(pep9) by exposing surface-immobilized ST(cys+)_(pep9) to a complex biological solution containing small concentrations of the target protein CDK2.     

Aptamer-based detection and quantitative analysis of ricin using affinity probe capillary electrophoresis

The ability to detect sub-nanomolar concentrations of ricin using fluorescently tagged RNA aptamers is demonstrated. Aptamers rival the specificity of antibodies and have the power to simplify immunoassays using capillary electrophoresis. Under nonequilibrium conditions, a dissociation constant, Kd, of 134 nM has been monitored between the RNA aptamer and ricin A-chain. With use of this free-solution assay, the detection of 500 pM (approximately 14 ng/mL) or 7.1 amol of ricin is demonstrated. The presence of interfering proteins such as bovine serum albumin and casein do not inhibit this interaction at sub-nanomolar concentrations. When spiked with RNAse A, ricin can still be detected down to 1 nM concentrations despite severe aptamer degradation. This approach offers a promising method for the rapid, selective, and sensitive detection of biowarfare agents.     

Multisegment nanowire sensors for the detection of DNA molecules

We describe a novel application for detecting specific single strand DNA sequences using multisegment nanowires via a straightforward surface functionalization method. Nanowires comprising CdTe-Au-CdTe segments are fabricated using electrochemical deposition, and electrical characterization indicates a p-type behavior for the multisegment nanostructures, in a back-to-back Schottky diode configuration. Such nanostructures modified with thiol-terminated probe DNA fragments could function as high fidelity sensors for biomolecules at very low concentration. The gold segment is utilized for functionalization and binding of single strand DNA (ssDNA) fragments while the CdTe segments at both ends serve to modulate the equilibrium Fermi level of the heterojunction device upon hybridization of the complementary DNA fragments (cDNA) to the ssDNA over the Au segment. Employing such multisegment nanowires could lead to the fabrication more sophisticated and high multispecificity biosensors via selective functionalization of individual segments for biowarfare sensing and medical diagnostics applications.     

Femtosecond resonance enhanced CARS for background-free detection of organic molecules

We study the coherent excitation profile (CEP) of resonance enhanced femtosecond CARS in a model system zinc phthalocyanine in a polymer film host as a prospective technique for detection and identification of molecular species in ambient environments. A new method of suppressing the non-resonant FWM background is demonstrated. Transform theory is applied to calculate CEP based on the absorption spectrum, and good agreement between theory and experiment is obtained.     

Biosensing systems for the detection of bacterial quorum signaling molecules

Bacterial quorum sensing (QS) is a cell-to-cell communication phenomenon that allows bacteria to control the expression of certain specialized genes depending on their cell population size. Signaling molecules such N-acylhomoserine lactones (AHLs) mediate the communication, and their concentration reflects the bacterial population density. Quorum sensing regulates several processes including bacterial pathogenicity. We developed a method for the rapid, sensitive, and quantitative detection of AHLs in biological samples such as saliva and stools. The method is based on whole-cell sensing systems that employ QS regulatory systems as recognition elements and the luxCDABE gene cassette as a reporter. The method proved to be reproducible when applied to real samples and was able to detect low analyte concentrations down to 1 x 10(-9) M without requiring extensive sample preparation. We envision that this novel biosensing system could be employed in the diagnosis and management of various bacteria-related disorders, thus supporting the use of quorum sensing molecules as potential biomarkers of disease. Due to cost-effectiveness and high throughput, these biosensing systems could be successfully employed as a new tool for the screening of novel drugs that target quorum sensing mechanisms.     

A hybrid plasmonic-photonic nanodevice for label-free detection of a few molecules

Noble metal nanowaveguides supporting plasmon polariton modes are able to localize the optical fields at nanometer level for high sensitivity biochemical sensing devices. Here we report on the design and fabrication of a novel photonic-plasmonic device which demonstrates label-free detection capabilities on single inorganic nanoparticles and on monolayers of organic compounds. In any case, we determine the Raman scattering signal enhancement and the device detection limits that reach a number of molecules between 10 and 250. The device can be straightforwardly integrated in a scanning probe apparatus with the possibility to match topographic and label-free spectroscopic information in a wide range of geometries.     

UV excitation thermal lens microscope for sensitive and nonlabeled detection of nonfluorescent molecules

An ultrasensitive and nonlabeled detection method of nonfluorescent molecules on a microchip was developed by realizing a thermal lens microscope (TLM) with a 266-nm UV pulsed laser as an excitation light source (UV-TLM). Pulsed laser sources have advantages over continuous-wave laser sources in more compact size and better wavelength tuning, which are important for microchip-based analytical systems. Their disadvantage is difficulty in applying a lock-in amplifier due to the high (>10(4)) duty ratio of pulse oscillation. To overcome this problem, we realized a quasi-continuous-wave excitation by modulating the pulse trains at approximately 1 kHz and detecting the synchronous signal with a lock-in amplifier. The optimum pulse repetition frequency was obtained at 80 kHz, which was reasonable considering thermal equilibrium time. Furthermore, a permissible flow velocity in the range of 6.6-19.8 mm/s was found to avoid sensitivity decrease due to photochemical reactions and thermal energy dissipation. Under these conditions, we detected adenine aqueous solutions on a fused-silica microchip without labeling and obtained a sensitivity that was 350 times higher than that in a spectrophotometric method. The sensitivity was enough for detection on a microchip with an optical path length that was 2-3 orders shorter than that in conventional cuvettes. Finally, the UV-TLM method was applied to liquid chromatography detection. Fluorene and pyrene were separated in a microcolumn and detected in a capillary (50-microm inner diameter) with 150 times higher sensitivity than a spectrophotometric method. Our method provides highly sensitive and widely applicable detections for various analytical procedures and chemical syntheses on microchips.     

Spin-stretching of DNA and protein molecules for detection by fluorescence and atomic force microscopy

We have developed a rapid and efficient way of stretching DNA and denatured protein molecules for detection by fluorescence microscopy and atomic force microscopy (AFM). In the described method, a viscous drag created by transient rotational flow stretches randomly coiled DNA molecules or denatured proteins. Stretching is achieved by dispensing a droplet of sample solution containing DNA or denatured protein on a MgCl2-soaked mica surface. We present fluorescent images of straightened lambdaDNA molecules and AFM images of stress-shared, reduced von Willebrand factor as well as straightened lambdaDNA. The described quick and reliable spin-stretching technique will find wide applications in the analysis of single biopolymer molecules.     

Porous silica as host for PEG-supported coumarin molecules

The design of novel organic–inorganic systems made of poly(ethyleneglycol) (PEG)-supported coumarin loaded into porous silica is presented. The hybrid system was obtained by impregnation of a xerogel silica matrix with a PEG-supported coumarin ethanolic solution. The chemical and physical properties of the hybrid systems are evaluated by means of infrared spectroscopy and thermo-gravimetric analysis, and correlated to UV–Vis optical absorption and time-resolved photoluminescence. It was found that the optical properties of the coumarin, which are not affected by the polymeric PEG support, are preserved upon loading into the silica porous support. The hybrid material obtained may represent a tool for drug delivery as the release of the PEG-supported coumarin from the silica xerogel into water media takes place.     

Broad-specificity immunoassays for sulfonamide detection: immunochemical strategy for generic antibodies and competitors

Development of antibodies with broad specificity recognition for sulfonamide drugs was found to be surprisingly difficult when conventional immunochemical strategies were applied to hapten design. To improve the cross-reactivity pattern of antibodies for the family of sulfonamide drugs, a novel strategy based on the single-ring (fragment-derived) hapten moieties with different spacer substituent lengths was employed for the preparation of immunogens, coating conjugates, and enzyme competitors. The rabbit antibodies raised against a common (one-ring) p-aminobenzenesulfonamide hapten moiety (attached to a carrier protein through the N-1 position) in combination with a homologous hapten-peroxidase tracer allowed the detection of 15 sulfonamide species at the maximum residue limit level using direct ELISA. The two-ring 6-(4-aminobenzensulfonylamino)hexanoic hapten mimics, previously reported in the literature as a weak generic antigen, generated surprisingly superior immune responses in rabbits. The antibodies raised against this two-ring hapten were capable of detecting at least 19 and 17 sulfonamides in a direct ELISA system at the regulatory level with sensitivities corresponding to 20 and 50% binding inhibition, respectively. A negligible cross-reaction with N4 metabolites makes it possible to measure responses of parent sulfonamides in the presence of their metabolized forms. In skimmed milk, the highest limit of detection (LOD) for sulfacetamide defined as 20% inhibition was 65.2 microg x L(-1) (IC20 value), whereas the additional 18 sulfonamides tested exhibited LODs in the range of 0.2-36.8 microg x L(-1). This sensitivity allows simple multisulfonamide tests to be established for use in the laboratory or on site.     

Simultaneous detection of single molecules and singulated ensembles of molecules enables immunoassays with broad dynamic range

We report a method for combining the detection of single molecules (digital) and an ensemble of molecules (analog) that is capable of detecting enzyme label from 10(-19) M to 10(-13) M, for use in high sensitivity enzyme-linked immunosorbent assays (ELISA). The approach works by capturing proteins on microscopic beads, labeling the proteins with enzymes using a conventional multistep immunosandwich approach, isolating the beads in an array of 50-femtoliter wells (Single Molecule Array, SiMoA), and detecting bead-associated enzymatic activity using fluorescence imaging. At low concentrations of proteins, when the ratio of enzyme labels to beads is less than ～1.2, beads carry either zero or low numbers of enzymes, and protein concentration is quantified by counting the presence of "on" or "off" beads (digital regime). (1) At higher protein concentrations, each bead typically carries multiple enzyme labels, and the average number of enzyme labels present on each bead is quantified from a measure of the average fluorescence intensity (analog regime). Both the digital and analog concentration ranges are quantified by a common unit, namely, average number of enzyme labels per bead (AEB). By combining digital and analog detection of singulated beads, a linear dynamic range of over 6 orders of magnitude to enzyme label was achieved. Using this approach, an immunoassay for prostate specific antigen (PSA) was developed. The combined digital and analog PSA assay provided linear response over approximately four logs of concentration ([PSA] from 8 fg/mL to 100 pg/mL or 250 aM to 3.3 pM). This approach extends the dynamic range of ELISA from picomolar levels down to subfemtomolar levels in a single measurement.     

Comparison of Hadamard transform and signal-averaged detection for microchannel electrophoresis

A Hadamard transform (HT) detection method for microchip capillary electrophoresis with laser-induced fluorescence and a charge-coupled device (CCD) is described and compared to signal-averaged detection. A low-noise CCD camera is used to image a section of a separation channel where each camera pixel can be thought of as a unique detector. For signal averaging, electropherograms corresponding to individual pixels can be averaged for improved S/N. HT detection is performed on each pixel electropherogram to generate a contour plot electropherogram. The multiple injections required for HT provides an enhancement at the cost of longer times for the pseudorandom injection sequences. A short sample injection length of 0.25 s is used to reduce the overall analysis time and improve sensitivity compared to previously published results. An injection sequence is performed on the microchip that is based on a cyclic S-matrix of 513 elements that generates an 8-fold improvement in S/N compared to a single injection. This spatially resolved HT detection method is also capable of performing a multicomponent separation. Signal-averaged HT and single-injection data are compared to experimental HT and single-injection results. The unique capabilities of each method are described.     

Forensic identification of neat ricin and of ricin from crude castor bean extracts by mass spectrometry

The protein toxin ricin, which originates from the seeds of Ricinus communis plants, has been the subject of increased interest, due to its potential terrorist use. Exceptionally, this toxin is also subject to the Chemical Weapons Convention. In this paper, it is shown that mass spectrometry can be used to unambiguously verify the presence of ricin in crude toxin preparations. It is demonstrated that MALDI MS can be used for screening, either by direct analysis or by trypsin digestion and peptide mapping. Purified ricin from several varieties of R. communis was characterized by LC-ES MS(/MS). A crude ricin preparation from a single bean was similarly characterized. An LC method was set up with product ion MS/MS detection of selected marker peptides specific for ricin: T5, T7, T11, T12, and T13 from the A-chain and T3, T5, T14, T19, and T20 from the B-chain. This method was then used to unambiguously identify ricin in a crude preparation of ricin. The MALDI MS molecular weight analysis and the marker peptides LC-ES MS/MS analysis give a forensic level of identification of ricin when combined with activity testing.     

Ultrasensitive coincidence fluorescence detection of single DNA molecules

We have detected individual DNA molecules labeled with two different fluorophores in solution by using two-color excitation and detection of coincidence fluorescence bursts. The confocal volumes of the two excitation lasers were carefully matched so that the volume overlap was 30% of the total confocal volume illuminated. This method greatly reduces the level of background fluorescence and, hence, extends the sensitivity of single molecule detection down to 50 fM. At these concentrations, the dual-labeled DNA is detectable in the presence of a 1000-fold excess of single-fluorophore-labeled DNA. We demonstrate that we can detect 100 fM dual-labeled DNA diluted in 1 microM unlabeled DNA, which was not possible with single color detection. This method can be used to detect rare molecules in complex mixtures.     

Focused ion beam-fabricated Au micro/nanostructures used as a surface enhanced Raman scattering-active substrate for trace detection of molecules and influenza virus

The focused ion beam (FIB) technique was used to precisely fabricate patterned Au micro/nanostructures (fibAu). The effects of surface enhanced Raman scattering (SERS) on the fibAu samples were investigated by adjusting the geometrical, dimensional, and spacing factors. The SERS mechanism was evaluated using low-concentration rhodamine 6G (R6G) molecules, physically adsorbed or suspended on/within the micro/nanostructures. The results indicated that for detecting R6G molecules, hexagon-like micro/nanostructures induced a higher electromagnetic mechanism (EM) due to the availability of multiple edges and small curvature. By decreasing the dimensions from 300 to 150 nm, the laser-focused area contained an increasing number of micro/nanostructures and therefore intensified the excitation of SERS signals. Moreover, with an optimized geometry and dimensions of the micro/nanostructures, the relative intensity/surface area value reached a maximum as the spacing was 22 nm. An exponential decrease was found as the spacing was increased, which most probably resulted from the loss of EM. The spacing between the micro/nanostructures upon the fibAu was consequently regarded as the dominant factor for the detection of R6G molecules. By taking an optimized fibAu to detect low-concentration influenza virus, the amino acids from the outermost surface of the virus can be well distinguished through the SERS mechanism.     

Metallic nanocone array photonic substrate for high-uniformity surface deposition and optical detection of small molecules

Molecular probe arrays printed on solid surfaces such as DNA, peptide, and protein microarrays are widely used in chemical and biomedical applications especially genomic and proteomic studies (Pollack et al 1999 Nat. Genet. 23 41-6, Houseman et al 2002 Nat. Biotechnol. 20 270-4, Sauer et al 2005 Nat. Rev. Genet. 6 465-76) as well as surface imaging and spectroscopy (Mori et al 2008 Anal. Biochem. 375 223-31, Liu et al 2006 Nat. Nanotechnol. 1 47-52, Liu 2010 IEEE J. Sel. Top. Quantum Electron. 16 662-71). Unfortunately the printed molecular spots on solid surfaces often suffer low distribution uniformity due to the lingering 'coffee stain' (Deegan et al 1997 Nature 389 827-9) problem of molecular accumulations and blotches, especially around the edge of deposition spots caused by solvent evaporation and convection processes. Here we present, without any surface chemistry modification, a unique solid surface of high-aspect-ratio silver-coated silicon nanocone arrays that allows highly uniform molecular deposition and thus subsequent uniform optical imaging and spectroscopic molecular detection. Both fluorescent Rhodamine dye molecules and unlabeled oligopeptides are printed on the metallic nanocone photonic substrate surface as circular spot arrays. In comparison with the printed results on ordinary glass slides and silver-coated glass slides, not only high printing density but uniform molecular distribution in every deposited spot is achieved. The high-uniformity and repeatability of molecular depositions on the 'coffee stain'-free nanocone surface is confirmed by laser scanning fluorescence imaging and surface enhanced Raman imaging experiments. The physical mechanism for the uniform molecular deposition is attributed to the superhydrophobicity and localized pinned liquid-solid-air interface on the silver-coated silicon nanocone surface. The unique surface properties of the presented nanocone surface enabled high-density, high-uniformity probe spotting beneficial for genomic and proteomic microarrays and surface molecular imaging.     

Noncompetitive phage anti-immunocomplex real-time polymerase chain reaction for sensitive detection of small molecules

Immuno polymerase chain reaction (IPCR) is an analytical technology based on the excellent affinity and specificity of antibodies combined with the powerful signal amplification of polymerase chain reaction (PCR), providing superior sensitivity to classical immunoassays. Here we present a novel type of IPCR termed phage anti-immunocomplex assay real-time PCR (PHAIA-PCR) for the detection of small molecules. Our method utilizes a phage anti-immunocomplex assay (PHAIA) technology in which a short peptide loop displayed on the surface of the M13 bacteriophage binds specifically to the antibody-analyte complex, allowing the noncompetitive detection of small analytes. The phagemid DNA encoding this peptide can be amplified by PCR, and thus, this method eliminates hapten functionalization or bioconjugation of a DNA template while providing improved sensitivity. As a proof of concept, two PHAIA-PCRs were developed for the detection of 3-phenoxybenzoic acid, a major urinary metabolite of some pyrethroid insecticides, and molinate, a herbicide implicated in fish kills. Our results demonstrate that phage DNA can be a versatile material for IPCR development, enabling universal amplification when the common element of the phagemid is targeted or specific amplification when the real time PCR probe is designed to anneal the DNA encoding the peptide. The PHAIA-PCRs proved to be 10-fold more sensitive than conventional PHAIA and significantly faster using magnetic beads for rapid separation of reactants. The assay was validated with both agricultural drain water and human urine samples, showing its robustness for rapid monitoring of human exposure or environmental contamination.     

Specific chemical effects on surface-enhanced Raman spectroscopy for ultra-sensitive detection of biological molecules

Achieving high signal amplification in surface-enhanced Raman scattering (SERS) is important for reaching single molecule level sensitivity and has been the focus of intense research efforts. We introduce a novel chemical enhancer, lithium chloride, that provides an additional order of magnitude increase in SERS relative to previously reported enhancement results. We have duplicated single molecule detection of the DNA base adenine that has previously been reported, thereby providing independent validation of this important result. Building upon this work, we show that the chemical enhancer LiCl produces strong SERS signal under a wide range of experimental conditions, including multiple laser excitation wavelengths and target molecule concentrations, for nucleotides, nucleosides, bases, and dye molecules. This is significant because while selection of anions used in chemical enhancement is well known to affect the degree of amplification attained, cation selection has previously been reported to have no major effect on the magnitude of SERS enhancement. Our findings indicate that cation selection is quite important in ultra-sensitive SERS detection, opening the door to further discussion and theory development involving the role of cations in SERS.     

Single-stranded DNA binding protein-assisted fluorescence polarization aptamer assay for detection of small molecules

Here, we describe a new fluorescence polarization aptamer assay (FPAA) strategy which is based on the use of the single-stranded DNA binding (SSB) protein from Escherichia coli as a strong FP signal enhancer tool. This approach relied on the unique ability of the SSB protein to bind the nucleic acid aptamer in its free state but not in its target-bound folded one. Such a feature was exploited by using the antiadenosine (Ade)-DNA aptamer (Apt-A) as a model functional nucleic acid. Two fluorophores (fluorescein and Texas Red) were introduced into different sites of Apt-A to design a dozen fluorescent tracers. In the absence of the Ade target, the binding of the labeled aptamers to SSB governed a very high fluorescence anisotropy increase (in the 0.130-0.200 range) as the consequence of (i) the large global diffusion difference between the free and SSB-bound tracers and (ii) the restricted movement of the dye in the SSB-bound state. When the analyte was introduced into the reaction system, the formation of the folded tertiary structure of the Ade-Apt-A complex triggered the release of the labeled nucleic acids from the protein, leading to a strong decrease in the fluorescence anisotropy. The key factors involved in the fluorescence anisotropy change were considered through the development of a competitive displacement model, and the optimal tracer candidate was selected for the Ade assay under buffer and realistic (diluted human serum) conditions. The SSB-assisted principle was found to operate also with another aptamer system, i.e., the antiargininamide DNA aptamer, and a different biosensing configuration, i.e., the sandwich-like design, suggesting the broad usefulness of the present approach. This sensing platform allowed generation of a fluorescence anisotropy signal for aptamer probes which did not operate under the direct format and greatly improved the assay response relative to that of the most previously reported small target FPAA.