NAGase sensing in 3% milk: FET-based specific and label-free sensing in ultra-small samples of high ionic strength and high concentration of non-specific proteins.

Biosensing with biological field-effect transistors (bioFETs) is a promising technology toward specific, label-free, and multiplexed sensing in ultra-small samples. The current study employs the field-effect meta-nano-channel biosensor (MNC biosensor) for the detection of the enzyme N-acetyl-beta-D-glucosaminidase (NAGase), a biomarker for milk cow infections. The measurements are performed in a 0.5 µL drops of 3% commercial milk spiked with NAGase concentrations in the range of 30.3 aM-3.03 µM (Note that there is no background NAGase concentration in commercial milk). Specific and label-free sensing of NAGase is demonstrated with a limit-of-detection of 30.3 aM, a dynamic range of 11 orders of magnitude and with excellent linearity and sensitivity. Additional two important research outcomes are reported. First, the ionic strength of the examined milk is ∼120 mM which implies a bulk Debye screening length <1 nm. Conventionally, a 1 nm Debye length excludes the possibility of sensing with a recognition layer composed of surface bound anti-NAGase antibodies with a size of ∼10 nm. This apparent contradiction is removed considering the ample literature reporting antibody adsorption in a predominantly surface tilted configuration (side-on, flat-on, etc.). Secondly, milk contains a non-specific background protein concentration of 33 mg/ml, in addition to considerable amounts of micron-size heterogeneous fat structures. The reported sensing was performed without the customarily exercised surface blocking and without washing of the non-specific signal. This suggests that the role of non-specific adsorption to the BioFET sensing signal needs to be further evaluated. Control measurements are reported.

From sensing interactions to controlling the interactions: a novel approach to obtain biological transistors for specific and label-free immunosensing.

Antibody-antigen interactions are shaped by the solution pH level, ionic strength, and electric fields, if present. In biological field-effect transistors (BioFETs), the interactions take place at the sensing area in which the pH level, ionic strength and electric fields are determined by the Poisson-Boltzmann equation and the boundary conditions at the solid-solution interface and the potential applied at the solution electrode. The present study demonstrates how a BioFET solution electrode potential affects the sensing area double layer pH level, ionic strength, and electric fields and in this way shapes the biological interactions at the sensing area. We refer to this as 'active sensing'. To this end, we employed the meta-nano-channel (MNC) BioFET and demonstrate how the solution electrode can determine the antibody-antigen equilibrium constant and allows the control and tuning of the sensing performance in terms of the dynamic range and limit-of-detection. In the current work, we employed this method to demonstrate the specific and label-free sensing of Alpha-Fetoprotein (AFP) molecules from 0.5 µL drops of 1 : 100 diluted serum. AFP was measured during pregnancy as part of the prenatal screening program for fetal anomalies, chromosomal abnormalities, and abnormal placentation. We demonstrate AFP sensing with a limit-of-detection of 10.5 aM and a dynamic range of 6 orders of magnitude in concentration. Extensive control measurements are reported.

Non-Contact, Continuous Sampling of Porous Surfaces for the Detection of Particulate and Adsorbed Organic Contaminations by Low-Temperature Plasma Coupled to Ion Mobility Spectrometer.

Chemical analysis of hazardous surface contaminations, such as hazardous substances, explosives or illicit drugs, is an essential task in security, environmental and safety applications. This task is mostly based on the collection of particles with swabs, followed by thermal desorption into a vapor analyzer, usually a detector based on ion mobility spectrometry (IMS). While this methodology is well established for several civil applications, such as border control, it is still not efficient enough for various conditions, as in sampling rough and porous surfaces. Additionally, the process of thermal desorption is energetically inefficient, requires bulky hardware and introduces device contamination memory effects. Low-temperature plasma (LTP) has been demonstrated as an ionization and desorption source for sample preparation-free analysis, mostly at the inlet of a mass spectrometer analyzer, and in rare cases in conjunction with an ion mobility spectrometer. Herein, we demonstrate, for the first time, the operation of a simple, low cost, home-built LTP apparatus for desorbing non-volatile analytes from various porous surfaces into the inlet of a handheld IMS vapor analyzer. We show ion mobility spectra that originate from operating the LTP jet on porous surfaces such as asphalt and shoes, contaminated with model amine-containing organic compounds. The spectra are in good correlation with spectra measured for thermally desorbed species. We verify through LC-MS analysis of the collected vapors that the sampled species are not fragmented, and can thus be identified by commercial IMS detectors.

A new approach towards the Debye length challenge for specific and label-free biological sensing based on field-effect transistors.

Biologically-modified field-effect transistors (BioFETs) are promising platforms for specific and label-free biosensing due to their sub-micron footprint suitable for multiplexing in ultra-small samples, low noise levels, inherent amplification, etc. Debye screening length is a well-recognized challenge for any BioFET-based technology. The screening length is the smallest at the double layer, where the solution ion population is higher than the bulk population. One way to address the small double layer screening length is to electrostatically modify the potential drop across the solution such as to minimize the potential drop over the double layer. This will decrease the population of the double layer ions and increase the screening length. However, this is not possible with BioFETs as voltage application to the reference electrode simultaneously affects both the double layer and the BioFET conducting channel. The current study addresses the screening length challenge with the novel Meta-Nano-Channel (MNC) BioFET. The MNC BioFET, which is fabricated in a complementary-metal-oxide-silicon (CMOS) process, allows decoupling of the electrostatics of the double layer from the electrodynamics of the conducting channel. The study explores the mechanism of sensing with the MNC BioFET, and demonstrates how the double layer can be electrostatically tuned in order to optimize the screening length without affecting the conducting channel. Finally, specific and label-free sensing of 10 ng ml-1 prostate specific antigen (PSA) is demonstrated. It is shown that by electrostatically increasing the double layer screening length, the sensing signal increases from 70 mV to 133 mV.

Sonic-spray introduction of liquid samples to hand-held Ion mobility spectrometry analyzers.

Sampling hazardous compounds in the form of solids and liquids is a growing need in the fields of homeland security and forensics. Chemical analysis of particles and droplets under field conditions is crucial for various tasks carried out by counter-terrorism and law enforcement units. The use of simple, small and low cost means to achieve this goal is constantly pursued. In this work, an approach for rapid, continuous generation of vapors from liquid samples using sonic spray (SS) as the sample introduction technique, followed by analysis using hand-held ion mobility spectrometry (IMS) vapor analyzers is presented. Transfer of analytes is demonstrated from liquid state to the gas phase at the inlet of an IMS detector using a sonic spray apparatus that consists of a nebulizer, spraying solution, a source of compressed gas and an unheated transfer line tube to the detector inlet nozzle. This technique does not require any electrical, radiative or thermal energy. Analysis of several narcotic substances including cocaine, methamphetamine and amphetamine, and of an explosive compound, TNT, is demonstrated, using two commercial devices as analyzers. Two sampling configurations are presented: direct sampling of liquid, either from a vial or a spill (SS-IMS) and extraction of a substance collected with a swab by dipping it in the spray solvent (ESS-IMS), being suitable for both drops and particles. Limits of detection of the presented method are comparable to those obtained with thermal desorption sample introduction of the commercial device. Time traces of the IMS signals show a continuous and stable signal with a short rise time. This sampling technique may offer competitive performance to that of common thermal desorption techniques, with the advantages of coupling to simpler, smaller and cheaper vapor detectors, optimized for field use, and of a continuous, pulseless sample or object interrogation.

Explosive vapour/particles detection using SERS substrates and a hand-held Raman detector.

We developed and optimized surface-enhanced Raman spectrometry (SERS) methods for trace analysis of explosive vapour and particles using a hand-held Raman spectrometer in the field. At first, limits of detection (LODs) using SERS methods based on a colloidal suspension of gold nanoparticles were measured under alkaline conditions and are as follows: pentaerythritol tetranitrate (PETN) (1.5 × 10-6 M, 6.9 ng), 1,3,5,7-tetranitro-1,3,5,7-tetrazoctane (HMX), 8.1 × 10-6 M, 35 ng; urea nitrate (UN), 9.2 × 10-4 M, 165 ng; 2,4,6-trinitrotoluene (TNT), 1.1 × 10-7 M, 0.35 ng. We developed SERS substrates that demonstrate the wide applicability of this technique for use in the field for explosive vapour and particles adsorbed on a surface based on Au nanoparticles that were optimal for the detection of the target materials in solution. Au nanoparticles were modified onto quartz fibres or a polyurethane sponge for vapour/particles detection. SERS detection of vapours of 2,4-dinitrotoluene (2,4-DNT) and 1,3-dinitrobenzene (1,3-DNB) was shown by sampling vapours onto Au-modified quartz fibres followed by hand-held Raman analysis with estimated minimum detection levels of 3.6 ng and 54 ng, respectively. The detection of 2,4-DNT using sponge-based SERS decorated with Au nanoparticles was also demonstrated; however, the sensitivity was lower than that observed using quartz fibres. The detection of TNT on a surface was performed by utilizing quartz-fibres precoated with alumina and modified with Au nanoparticles, and the detection of 10 µg (0.53 µg cm-2) of TNT was demonstrated.

Surface-enhanced Raman spectroscopy (SERS) for detection of VX and HD in the gas phase using a hand-held Raman spectrometer.

A sensitive surface-enhanced Raman spectroscopy (SERS) substrate was developed to enable hand-held Raman spectrometers to detect gas-phase VX and HD. The substrate comprised Au nanoparticles modified onto quartz fibres. Limits of detection (LOD) of 0.008 µg L-1 and 0.054 µg L-1 were achieved for VX and HD, respectively. Gas-phase experiments were performed using a homemade gas-phase flow system inside a climatic chamber at 25 °C and 50% relative humidity. Preliminary experiments were conducted using VX and HD in solution with Au and Ag nanoparticle colloidal suspensions. We developed and optimized several SERS methods for detection of VX and HD in solution. Gold nanoparticles were optimal for detection of VX and HD and were modified onto quartz fibres for gas-phase detection. The LODs for HD and VX detection in solution were 1.8 × 10-3 µg mL-1 (1.1 × 10-8 M) and 2.5 × 10-3 µg mL-1 (9.3 × 10-9 M), respectively. This study demonstrates that integration of SERS substrates with hand-held Raman spectrometers expands the applicability of Raman technology to homeland security, as reflected by increased sensitivity and gas-phase detection capabilities.

Non-contact and non-destructive detection and identification of Bacillus anthracis inside paper envelopes.

Efficient and safe detection of Bacillus anthracis spores (BAS) is a challenging task especially in bio-terror scenarios where the agent is concealed. We provide a proof-of-concept for the identification of concealed BAS inside mail envelopes using short-wave infrared hyperspectral imaging (SWIR-HSI). The spores and two other benign materials are identified according to their typical absorption spectrum. The identification process is based on the removal of the envelope signal using a new automatic new algorithm. This method may serve as a fast screening tool prior to using classical bioanalytical techniques.

A Rapid Antimicrobial Susceptibility Test for Determining Yersinia pestis Susceptibility to Doxycycline by RT-PCR Quantification of RNA Markers.

Great efforts are being made to develop new rapid antibiotic susceptibility tests to meet the demand for clinical relevance versus disease progression. This is important especially in diseases caused by bacteria such as Yersinia pestis, the causative agent of plague, which grows rapidly in vivo but relatively slow in vitro. This compromises the ability to use standard growth-based susceptibility tests to obtain rapid and proper antibiotic treatment guidance. Using our previously described platform of quantifying antibiotic-specific transcriptional changes, we developed a molecular test based on changes in expression levels of doxycycline response-dependent marker genes that we identified by transcriptomic analysis. This enabled us to determine the minimal inhibitory concentration of doxycycline within 7 h compared to the 24 h required by the standard CLSI test. This assay was validated with various Y. pestis strains. Moreover, we demonstrated the applicability of the molecular test, combined with a new rapid bacterial isolation step from blood cultures, and show its relevance as a rapid test in clinical settings.

Characterization of peptides self-assembly by low frequency Raman spectroscopy.

Low Frequency Vibrational (LFV) modes of peptides and proteins are attributed to the lattice vibrations and are dependent on their structural organization and self-assembly. Studies taken in order to assign specific absorption bands in the low frequency range to self-assembly behavior of peptides and proteins have been challenging. Here we used a single stage Low Frequency Raman (LF-Raman) spectrometer to study a series of diastereomeric analogue peptides to investigate the effect of peptides self-assembly on the LF-Raman modes. The structural variation of the diastereomeric analogues resulted in distinct self-assembly groups, as confirmed by transmission electron microscopy (TEM) and dynamic light scattering (DLS) data. Using LF-Raman spectroscopy, we consistently observed discrete peaks for each of the self-assembly groups. The correlation between the spectral features and structural morphologies was further supported by principal component analysis (PCA). The LFV modes provide further information on the degrees of freedom of the entire peptide within the higher order organization, reflecting the different arrangement of its hydrogen bonding and hydrophobic interactions. Thus, our approach provides a simple and robust complementary method to structural characterization of peptides assemblies.

Optimized data analysis algorithm for on-site chemical identification using a hand-held attenuated total reflection Fourier transform infrared (ATR FT-IR) spectrometer.

On-site identification of organic compounds in the presence of interfering materials using a field-portable attenuated total reflection Fourier transform infrared (ATR FT-IR) spectrometer is presented. Identification is based on an algorithm that compares the analyte's infrared absorption spectrum with the reference spectra. The comparison is performed at several predetermined frequencies, and a similarity value (distance) between the measured and the reference spectra is calculated either at each frequency individually, or, alternatively, the average distance for all frequencies is calculated. The examined frequencies are selected to give the best contrast between the target materials of interest. In this study, the algorithm was optimized to identify three common chemical warfare agents (CWAs): O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioic acid (VX), sarin (GB), and sulfur mustard (bis(2-chloroethyl) sulfide) (HD), in the presence of field-related interfering materials (fuels, water, and dust). Receiver operating characteristics analysis was performed in order to determine the probabilities for detection (PD) and for false alerts (PF). Challenging the algorithm with a set of data that contains mixtures of CWAs and interfering materials resulted in PD of 90% and PF of 0%, 0%, and 1% for VX, GB, and HD, respectively, using the average distance approach, which was found to be much more effective than analyzing each frequency individually. This finding was validated for all possible combinations of 2-7 peaks per material. It is suggested that this algorithm provides a reliable mean for the identification of a predetermined set of target analytes and interfering materials.

Proteins as electronic materials: electron transport through solid-state protein monolayer junctions.

Electron transfer (ET) through proteins, a fundamental element of many biochemical reactions, is studied intensively in aqueous solutions. Over the past decade, attempts were made to integrate proteins into solid-state junctions in order to study their electronic conductance properties. Most such studies to date were conducted with one or very few molecules in the junction, using scanning probe techniques. Here we present the high-yield, reproducible preparation of large-area monolayer junctions, assembled on a Si platform, of proteins of three different families: azurin (Az), a blue-copper ET protein, bacteriorhodopsin (bR), a membrane protein-chromophore complex with a proton pumping function, and bovine serum albumin (BSA). We achieve highly reproducible electrical current measurements with these three types of monolayers using appropriate top electrodes. Notably, the current-voltage (I-V) measurements on such junctions show relatively minor differences between Az and bR, even though the latter lacks any known ET function. Electron Transport (ETp) across both Az and bR is much more efficient than across BSA, but even for the latter the measured currents are higher than those through a monolayer of organic, C18 alkyl chains that is about half as wide, therefore suggesting transport mechanism(s) different from the often considered coherent mechanism. Our results show that the employed proteins maintain their conformation under these conditions. The relatively efficient ETp through these proteins opens up possibilities for using such biomolecules as current-carrying elements in solid-state electronic devices.

Proteins as solid-state electronic conductors.

Protein structures can facilitate long-range electron transfer in solution. But a fundamental question remains: can these structures also serve as solid-state electronic conductors? Answering this question requires methods for studying conductivity of the "dry" protein (which only contains tightly bound structured water molecules) sandwiched between two electronic conductors in a solid-state type configuration. If successful, such systems could serve as the basis for future, bioinspired electronic device technology. In this Account, we survey, analyze, and compare macroscopic and nanoscopic (scanning probe) solid-state conductivities of proteins, noting the inherent constraints of each of these, and provide the first status report on this research area. This analysis shows convincing evidence that "dry" proteins pass orders of magnitude higher currents than saturated molecules with comparable thickness and that proteins with known electrical activity show electronic conductivity, nearly comparable to that of conjugated molecules ("wires"). These findings suggest that the structural features of proteins must have elements that facilitate electronic conductivity, even if they do not have a known electron transfer function. As a result, proteins could serve not only as sensing, polar,or photoactive elements in devices (such as field-effect transistor configurations) but also as electronic conductors. Current knowledge of peptide synthesis and protein modification paves the way toward a greater understanding of how changes in a protein's structure affect its conductivity. Such an approach could minimize the need for biochemical cascades in systems such as enzyme-based circuits, which transduce the protein's response to electronic current. In addition, as precision and sensitivity of solid-state measurements increase, and as knowledge of the structure and function of "dry" proteins grows, electronic conductivity may become an additional approach to study electron transfer in proteins and solvent effects without the introduction of donor or acceptor moieties. We are particularly interested in whether evolution might have prompted the electronic carrier transport capabilities of proteins for which no electrically active function is known in their native biological environment and anticipate that further research may help address this fascinating question.

Selective electroless deposition of metal clusters on solid-supported bacteriorhodopsin: applications to orientation labeling and electrical contacts.

Controlling the orientation of bacteriorhodopsin (bR) monolayers is an important step in studying and utilizing such membranes in a solid-state configuration in, for example, photoelectric applications. Macroscopic monolayers of bR have been fabricated in a variety of ways, but characterization of the distribution of the two possible orientations in which the membrane fragments can adsorb has not yet been addressed experimentally. Here, an approach is presented that labels only one of the membrane surfaces by electroless growth of metal nanoparticles on top of the solid-supported membranes. In this way, it is possible to observe which surface of the membranes is actually adsorbed to the substrate. How this technique serves to interface the membranes with a top metal contact for further electrical measurements is also demonstrated.

Bacteriorhodopsin as an electronic conduction medium for biomolecular electronics.

Interfacing functional proteins with solid supports for device applications is a promising route to possible applications in bio-electronics, -sensors, and -optics. Various possible applications of bacteriorhodopsin (bR) have been explored and reviewed since the discovery of bR. This tutorial review discusses bR as a medium for biomolecular optoelectronics, emphasizing ways in which it can be interfaced, especially as a thin film, solid-state current-carrying electronic element.

Covalent attachment of bacteriorhodopsin monolayer to bromo-terminated solid supports: preparation, characterization, and protein stability.

The interfacing of functional proteins with solid supports and the study of related protein-adsorption behavior are promising and important for potential device applications. In this study, we describe the preparation of bacteriorhodopsin (bR) monolayers on Br-terminated solid supports through covalent attachment. The bonding, by chemical reaction of the exposed free amine groups of bR with the pendant Br group of the chemically modified solid surface, was confirmed both by negative AFM results obtained when acetylated bR (instead of native bR) was used as a control and by weak bands observed at around 1610 cm(-1) in the FTIR spectrum. The coverage of the resultant bR monolayer was significantly increased by changing the pH of the purple-membrane suspension from 9.2 to 6.8. Although bR, which is an exceptionally stable protein, showed a pronounced loss of its photoactivity in these bR monolayers, it retained full photoactivity after covalent binding to Br-terminated alkyls in solution. Several characterization methods, including atomic force microscopy (AFM), contact potential difference (CPD) measurements, and UV/Vis and Fourier transform infrared (FTIR) spectroscopy, verified that these bR monolayers behaved significantly different from native bR. Current-voltage (I-V) measurements (and optical absorption spectroscopy) suggest that the retinal chromophore is probably still present in the protein, whereas the UV/Vis spectrum suggests that it lacks the characteristic covalent protonated Schiff base linkage. This finding sheds light on the unique interactions of biomolecules with solid surfaces and may be significant for the design of protein-containing device structures.

Functional optical detection based on pH dependent fluorescence lifetime.

BACKGROUND AND OBJECTIVES: Detection of possible alterations of physiological parameters (e.g., pH and temperature), resulting from malignant transformation of initially healthy tissue, can be a powerful diagnostic tool for earlier cancer detection. Such variations can be observed by comparing these parameters with those of healthy tissue surrounding the abnormality. Time-resolved spectroscopy of specifically targeted fluorescent labeled antibodies can be sensitive to such variations and provide a high resolution functional image of the region of interest. The goal of this study was to establish a forward experimental setup for calibration of the lifetime dependencies of near-IR fluorescent dyes on physiological parameters, and to develop analytical solutions, taking into account the effects of light propagation in turbid media (e.g., tissue), that was able to extract an original lifetime fluorescence signal from time-of-flight intensity distributions, measured in vivo from a deeply embedded live organ for further analysis. STUDY DESIGN/MATERIALS AND METHODS: Tissue-like phantoms with embedded fluorescent dyes and background optical properties simulating those of live tissues were designed and created. Fluorescence decay curves were measured for different fluorophore positions, and pH values. Those measurements were made with a system based on a time-correlated single photon counting (TCSPC) instrument and a tunable femtosecond Ti-Sapphire system built by our group. RESULTS: Decay curves were recorded for fluorophore depths of up to 5 mm and source-detector separation of 7 mm. It was shown that a forward model, based on the random walk theory, adequately described the experimental data. Measured pH dependencies of the fluorescence lifetime were characterized for two different dyes. CONCLUSIONS: Good correlation between experimental data and predictions of the theoretical model allows the use of close-form analytical solutions to separate the effects of photon time delays due to multiple scattering in tissues from the original intensity fluorescence time decay curve, determined by the fluorophore itself and its immediate surroundings. It is the latter dependence that can be diagnostically important. Experimentally obtained scaling between lifetime and a parameter of interest can be used in vivo to obtain a map of physiological parameter changes which can serve as a base for an in vivo specific diagnostic system.