Locating a compact odor source using a four-channel insect electroantennogram sensor

Here we demonstrate the feasibility of using an array of live insects to detect concentrated packets of odor and infer the location of an odor source (～15 m away) using a backward Lagrangian dispersion model based on the Langevin equation. Bayesian inference allows uncertainty to be quantified, which is useful for robotic planning. The electroantennogram (EAG) is the biopotential developed between the tissue at the tip of an insect antenna and its base, which is due to the massed response of the olfactory receptor neurons to an odor stimulus. The EAG signal can carry tens of bits per second of information with a rise time as short as 12 ms (K A Justice 2005 J. Neurophiol. 93 2233-9). Here, instrumentation including a GPS with a digital compass and an ultrasonic 2D anemometer has been integrated with an EAG odor detection scheme, allowing the location of an odor source to be estimated by collecting data at several downwind locations. Bayesian inference in conjunction with a Lagrangian dispersion model, taking into account detection errors, has been implemented resulting in an estimate of the odor source location within 0.2 m of the actual location.     

Odor discrimination using insect electroantennogram responses from an insect antennal array

Insects have a highly developed olfactory sensory system, mainly based in their antennae, for the detection and discrimination of volatile compounds in the environment. Electroantennogram (EAG) response profiles of five different insect species, Drosophila melanogaster, Heliothis virescens, Helicoverpa zea, Ostrinia nubilalis and Microplitis croceipes, showed different, species-specific EAG response spectra to 20 volatile compounds tested. The EAG response profiles were then reconstructed for each compound across the five insect species. Most of the compounds could be distinguished by comparing the response spectra. We then used a four-antenna array, called a Quadro-probe EAG, to see if we could discriminate among odorants based on the relative EAG amplitudes evoked when the probe was placed in plumes in a wind tunnel and in a field. Stable EAG responses could be simultaneously and independently recorded with four different insect antennae mounted on the Quadro-probe, and different volatile compounds could be distinguished in real time by comparing relative EAG responses with a combination of differently tuned insect antennae. Regardless of insect species or EAG amplitudes, antennae on the Quadro-probe maintained their responsiveness with higher than 1 peak/s of time resolution.     

Improvement of signal-to-noise ratio in electroantennogram responses using multiple insect antennae

Using an array of insect antennae connected in series or in parallel, electroantennogram (EAG) responses and noise levels were investigated in an attempt to improve signal-to-noise (S/N) ratio and sensitivity. Both the EAG response amplitude and noise level increased when the antennae of male Helicoverpa zea (Lepidoptera: Noctuidae) were connected in series. Due to lower relative increase in noise level than EAG amplitude as the number of antennae increased, the S/N ratio was also significantly improved by the serial connection. As a result the sensitivity of EAG was improved by the serial connection, which showed ca. ten-fold improvement in the threshold detection levels compared with a single antenna when four antennae were connected in series. In contrast to the serial connection, there were no differences in EAG amplitudes and overall noise levels when different numbers of antennae were connected in parallel. When only large-amplitude noise was taken into account, however, the S/N ratio was somewhat improved by the parallel connection. The frequency of overall noise remained at the same level both in the serial and in the parallel connection. However, the frequency of the large-amplitude noise increased in serial connection but decreased in parallel connection. The present study clearly indicates that both the sensitivity and S/N ratio of the EAG biosensor could be significantly improved by using the multiple antennal connections.     

Disruption of GABAA in the insect antennal lobe generally increases odor detection and discrimination thresholds

Studies of olfactory function show that disruption of GABA A receptors within the insect antennal lobe (AL) disrupts discrimination of closely related odors, suggesting that local processing within the AL specifically enhances fine odor discrimination. It remains unclear, however, how extensively AL function has been disrupted in these circumstances. Here we psychophysically characterize the effect of GABA A blockade in the AL of the moth Manduca sexta. We used 2 GABA A antagonists and 3 Pavlovian-based behavioral assays of olfactory function. In all cases, we used matched saline-injected controls in a blind study. Using a stimulus generalization assay, we found that GABA A disruption abolished the differential response to related odors, suggesting that local processing mediates fine odor discrimination. We then assessed the effect of GABA A antagonist on discrimination thresholds. Moths were differentially conditioned to respond to one odor (reinforced conditioned stimulus [CS+]) but not a second (unreinforced conditioning stimulus [CS-]) then tested for a significant differential conditioned response between them across a series of increasing concentrations. Here, GABA A blockade disrupted discrimination of both similar and dissimilar odor pairs as indicated by generally increased discrimination thresholds. Finally, using a detection threshold assay, we established that GABA A blockade also increases detection thresholds. Because detection is a prerequisite of discrimination, this later finding suggests that disrupted discrimination may be due to impairment of the ability to detect. We conclude that the loss of ability to detect and subsequently discriminate is attributable to a loss of ability of the AL to provide a clear neural signal from background.     

The label free DNA sensor using a silicon nanowire array

Biosensors based on silicon nanowire (Si-NW) promise highly sensitive dynamic label free electrical detection of various biological molecules. Here we report Si-NW array electronic devices that function as sensitive and selective detectors of as synthesized 2D DNA lattices with biotins. The Si-NW array was fabricated using top-down approach consists of 250 nanowires of 20 μm in length, equally spaced with an interval of 3.2 μm. Measurements of photoresistivity of the Si-NW array device with streptavidin (SA) attached on biotinylated DNA lattices at different concentration were observed and analyzed.. The conductivity in the DNA lattices with protein SA shows significant change in the photoresistivity of Si-NW array device. This Si-NW based DNA sensor would be one of very efficient devices for direct, label free DNA detection and could provide a pathway to immunological assays, DNA forensics and toxin detection in modern biotechnology.     

Versatile bioelectronic interfaces based on heterotrifunctional linking molecules

Bioelectronic interfaces that allow dehydrogenase enzymes to communicate with electrodes have potential applications such as biosensors and biocatalytic reactors. A major challenge in creation of such bioelectronic interfaces is to orient the enzyme, its cofactor, and an electron mediator properly with respect to the electrode in order to achieve efficient, multistep electron transfer. This paper describes a versatile, new method that uses cysteine, an inexpensive, branched amino acid having sulfhydryl, amino, and carboxyl functional groups, to achieve such orientation. This approach provides greater flexibility in assembling complex bioelectronic interfaces than previously reported approaches that bind the enzyme, cofactor, and mediator in a linear chain. Cysteine was attached to a gold electrode through the sulfhydryl groups, to the electron mediator toluidine blue O (TBO) through the carboxyl group, and to the cofactor (e.g., NAD(P)+) through the amino group. Cyclic voltammetry, impedance spectroscopy, chronoamperometry and quartz crystal microbalance gravimetry were used to demonstrate the sequential assembly steps and the electrical activity of the resulting bioelectronic interface.     

High sensitivity carbon nanotube based electrochemiluminescence sensor array

Ink jet printed carbon nanotube forest arrays capable of detecting picomolar concentrations of immunoglobulin G (IgG) using electrochemiluminescence (ECL) are described. Patterned arrays of vertically aligned single walled carbon nanotube (SWCNT) forests were printed on indium tin oxide (ITO) electrodes. Capture anti-IgG antibodies were then coupled through peptide bond formation to acidic functional groups on the vertical nanotubes. IgG immunoassays were performed using silica nano particles (Si NP) functionalized with the ECL luminophore [Ru(bpy)(2)PICH(2)](2+)], and IgG labelled G1.5 acid terminated PAMAM dendrimers. PAMAM is poly(amido amine), bpy is 2,2'-bipyridyl and PICH(2) is (2-(4-carboxyphenyl)imidazo[4,5-f][1,10]phenanthroline). The carboxyl terminal of [Ru(bpy)(2)PICH(2)](2+) (fluorescence lifetime ≈ 682±5 ns) dye was covalently coupled to amine groups on the 800 nm diameter silica spheres in order to produce significant ECL enhancement in the presence of sodium oxalate as co-reactant in PBS at pH 7.2). Significantly, this SWCNT-based sensor array shows a wide linear dynamic range for IgG coated spheres (10(6) to 10(12) spheres) corresponding to IgG concentrations between 20 pM and 300 nM. A detection limit of 1.1±0.1 pM IgG is obtained under optimal conditions.     

Learned odor discrimination in Drosophila without combinatorial odor maps in the antennal lobe

A unifying feature of mammalian and insect olfactory systems is that olfactory sensory neurons (OSNs) expressing the same unique odorant-receptor gene converge onto the same glomeruli in the brain [1-7]. Most odorants activate a combination of receptors and thus distinct patterns of glomeruli, forming a proposed combinatorial spatial code that could support discrimination between a large number of odorants [8-11]. OSNs also exhibit odor-evoked responses with complex temporal dynamics [11], but the contribution of this activity to behavioral odor discrimination has received little attention [12]. Here, we investigated the importance of spatial encoding in the relatively simple Drosophila antennal lobe. We show that Drosophila can learn to discriminate between two odorants with one functional class of Or83b-expressing OSNs. Furthermore, these flies encode one odorant from a mixture and cross-adapt to odorants that activate the relevant OSN class, demonstrating that they discriminate odorants by using the same OSNs. Lastly, flies with a single class of Or83b-expressing OSNs recognize a specific odorant across a range of concentration, indicating that they encode odorant identity. Therefore, flies can distinguish odorants without discrete spatial codes in the antennal lobe, implying an important role for odorant-evoked temporal dynamics in behavioral odorant discrimination.     

Real-time detection of BRCA1 gene mutations using a monolithic silicon optocoupler array

The monolithic silicon optocoupler presented here offers a platform for a new generation of fully integrated devices fabricated through the mature and inexpensive silicon technology. Using the developed optocoupler, real-time detection of the most common mutations in BRCA1 gene related to predisposition to hereditary breast/ovarian cancer was accomplished. For this purpose, oligonucleotides corresponding to wild- and mutant-type sequences were immobilized onto different optocouplers and the hybridization with fluorescently labeled complementary or non-complementary sequences was monitored in real time. Hybridization of fluorescently labeled oligonucleotides to the immobilized ones modulated the coupling efficiency between the light emitting diode and the detector in a concentration-dependent manner. Using as label the AlexaFluor 647 dye (whose absorption maximum fits the emission maximum of the light source) a detection limit of 0.9 nM (9 fmol) was achieved. Real-time signal monitoring, especially during dehybridization, improved considerably the discrimination between wild-type and mutant sequences due to the ability to calculate dissociation kinetics upon washing independently for each one mutation. The bioanalytical capabilities of the transducer along with the fact that dense transducer arrays can be fabricated on a single chip open new frontiers in the manufacturing of microsystems appropriate for point-of-care analysis.     

Characterization of multicomponent monosaccharide solutions using an enzyme-based sensor array

We report the development of a sensor for rapidly and simultaneously measuring multiple sugars in aqueous samples. In this strategy, enzyme-based assays are localized within an array of individually addressable sites on a micromachined silicon chip. Microspheres derivatized with monosaccharide-specific dehydrogenases are distributed to pyramidal cavities anisotropically etched in a wafer of silicon (100) and are exposed to sample solution that is forced through the cavities by a liquid chromatography pumping system. Production of fluorescent reporter molecules is monitored under stopped-flow conditions when localized dehydrogenase enzyme systems are exposed to their target sugars. We demonstrate the capability of this analysis strategy to quantify beta-D-glucose and beta-D-galactose at low micromolar to millimolar levels, with no detectable cross-talk between assay sites. Analysis is achieved either through fluorescence detection of an initial dehydrogenase product (NADH, NADPH) or by production of a secondary fluorescent product created by hydride transfer from the reduced nicotinamide cofactor to a fluorogenic reagent. The array format of this sensor provides capabilities for redundant analysis of sugars and for monitoring levels of other solution components known to affect the activity of enzymes. The use of this strategy to normalize raw fluorescence signals is demonstrated by the determination of glucose and pH on a single chip. Alternatively, uncertainties in the activity of an immobilized enzyme can be accounted for using standard additions, an approach used here in the determination of serum glucose.     

Ligase-based multiple DNA analysis by using an electrochemical sensor array

We herein report an electrochemical biosensor for the sequence-specific detection of DNA with high discrimination ability for single-nucleotide polymorphisms (SNPs). This DNA sensor was constructed by a pair of flanking probes that "sandwiched" the target. A 16-electrode electrochemical sensor array was employed, each having one individual DNA capture probe immobilized at gold electrodes via gold-thiol chemistry. By coupling with a biotin-tagged detection probe, we were able to detect multiple DNA targets with a single array. In order to realize SNP detection, a ligase-based approach was employed. In this method, both the capture probe and the detection probe were in tandem upon being hybridized with the target. Importantly, we employed a ligase that specifically could ligate tandem sequences only in the absence of mismatches. As a result, when both probes were complementary to the target, they were ligated in the presence of the ligase, thus being retained at the surface during the subsequent stringent washing steps. In contrast, if there existed 1-base mismatch, which could be efficiently recognized by the ligase, the detection probe was not ligated and subsequently washed away. A conjugate of avidin-horseradish peroxidase was then attached to the biotin label at the end of the detection probe via the biotin-avidin bridge. We then electrochemically interrogated the electrical current for the peroxidase-catalyzed reduction of hydrogen peroxide. We demonstrated that the electrochemical signal for the wild-type DNA was significantly larger than that for the sequence harboring the SNP.     

An electrochemical sensor based on cellulose nanocrystal for the enantioselective discrimination of chiral amino acids

A novel electrochemical sensor based on 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized cellulose nanocrystals (TOCNCs) and l-cystines (l-Cys) modified Au electrode (TOCNC/l-Cys/Au) has been fabricated for detection and discrimination of the enantiomers of phenylalanine (Phe), leucine (Leu), and valine (Val). The three amino acids are in connection with metabolism diseases. The TOCNC/l-Cys/Au electrode exhibited obvious peak current difference for the amino acid enantiomers by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The TOCNCs on the electrode surface expressed different interactions with d- and l-amino acids, so the electrochemical recognitions of the three amino acid enantiomers were achieved. TOCNCs were characterized by Fourier transform infrared (FT-IR) and scanning electron microscopy (SEM). The modified electrodes were characterized by SEM and electrochemical techniques. According to DPV, peak currents of the two enantiomers decreased linearly with their concentrations. Furthermore, satisfactory results were obtained when this electrode was applied to measure the d- and l-Phe mixture. The experimental results show that TOCNCs are suitable material for chiral sensor. The contrast of serum sample of healthy people and patients with type 2 diabetes also was proposed, and significant difference was exhibited on the modified electrode. This work is significant for the screening, diagnosis, and treatment of multiple metabolic diseases.     

Label-free antibody-antigen binding detection by optical sensor array based on surface-synthesized gold nanoparticles

Gold nanoparticles grown in situ from printed seed particles on a glass substrate have been fabricated into a biosensor array. The light-scattering properties of the resulting surfaces show sensitivity to changes in the local refractive index. Each array spot is functionalized with fibrinogen or bovine serum albumin and scattered radiation is used to monitor the refractive index change on label-free binding of the antibodies to their antigens from whole blood antiserum. Data were collected real-time and the association rate constants for the specific antibody-antigen binding were derived from a kinetic analysis. The minimum antibody concentration detection sensitivity is of 100 nM.     

A neural mechanism of hierarchical discrimination of odors in the olfactory cortex based on spatiotemporal encoding of odor information

We propose a neural mechanism for discrimination of different complex odors in the olfactory cortex based on the dynamical encoding scheme. Both constituent molecules of the odor and their mixing ratios are encoded simultaneously into a spatiotemporal activity pattern (limit cycle attractor) in the olfactory bulb [Hoshino O, Kashimori Y, Kambara T (1998) Biol Cybern 79:109–120]. We present a functional model of the olfactory cortex consisting of some dynamical mapping modules. Each dynamical map is represented by itinerancy among the limit cycle attractors. When a temporal sequence of spatial activity patterns corresponding to a complex odor is injected from the bulb to the network of the olfactory cortex, the neural activity state of each mapping module is fixed to a relevant spatial pattern injected. Recognition of an odor is accomplished by a combination of firing patterns fixed in all the mapping modules. The stronger the response strength of the component, the earlier the component is recognized. The hierarchical discrimination of an odor is made by recognizing the components in order of decreasing response strengths. Received: 28 November 1998 / Accepted in revised form: 17 December 1999     

Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology.

We report here the development and application of a biosensor-based technology that employs surface plasmon resonance for label-free studies of molecular interactions in real time. The sensor chip interface, comprising a thin layer of gold deposited on a glass support, is derivatized with a flexible hydrophilic polymer to facilitate the attachment of specific ligands to the surface and to increase the dynamic range for surface concentration measurements. The sensor can be used to measure surface concentrations down to 10 pg/mm2. Typical coefficients of variation are from two to five percent. We anticipate that the ability to monitor multi-molecular complexes as they form will greatly contribute to the understanding of biorecognition and the structural basis of molecular function.     

Real-time detection of DNA hybridization on microarray using a CCD-based imaging system equipped with a rotated microlens array disk

This work describes a novel charge-coupled device (CCD)-based imaging system (MB Biochip Reader?) for real-time detection of DNA hybridization to DNA microarrays. The MB Biochip Reader? consisted of a laser light source (532 nm), a microlens array for generation of a multi-beam laser, and a CCD for 2-D signal imaging. The MB Biochip Reader? with a rotated microlens array, allowed large-field imaging (6.2 mm × 7.6 mm with 6.45 μm resolution) with fast time-resolution at 0.2 s without speckle noise. Furthermore, real-time detection of DNA hybridization, which is sufficient to obtain accurate data from tens of thousands of array element per field, was successfully performed without the need for laser scanning. The performance of the MB Biochip Reader? for DNA microarray imaging was similar to the commercially available photomultiplier tube (PMT)-based microarray scanner, ScanArray Lite. The system potentially could be applied toward real-time analysis in many other fluorescent techniques in addition to real-time DNA microarray analysis.     

Kin discrimination and altruism in the larvae of a solitary insect

Kin selection theory predicts altruism between related individuals, which requires the ability to recognize kin from non-kin. In insects, kin discrimination associated with altruistic behaviour is well-known in clonal and social species but in very few solitary insects. Here, we report that the solitary larvae of a non-social insect Aleochara bilineata Gyll. (Coleoptera; Staphylinidae) show kin discrimination and sibling-directed altruistic behaviour. Larvae superparasitize more frequently the hosts parasitized by non-kin individuals than those hosts parasitized by siblings. Kin discrimination probably occurs by self-referent phenotype matching, where an individual compares its own phenotype with that of a non-familiar related individual, a mechanism rarely demonstrated in animals. The label used to recognize kin from non-kin corresponds to substances contained in the plug placed on the hosts by the resident larvae during the parasitization process. Kin competition induced by a limited larval dispersion may have favoured the evolution of kin recognition in this solitary species.     

Following hybridization on sensor/array platforms by using SPR,elipsometer and MALDI-MS

Abstract

The aim of this study is to develop a methodology in which Surface Plasmon Resonance (SPR), Ellipsometer (EM) and Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI-MS) will be used together for detection of single-strand oligodeoxynucleotides (ssODNs) targets. A selected target-ssODNs, and its complementary, the probe-ssODNs carrying a -SH end group, a spacer arm (HS-(CH₂)₆–(T)₁₅, and a non-complementary ssODNs were used. Silicone based stamps with 16 regions were prepared and used for micro-contact printing (µCP) of the probe-ssODNs on the gold coated surfaces homogeneously. A modulator-spacer molecule (6-mercapto-1-hexanol) was co-immobilized to control surface probe density, to orientate the probe-ssODNs, and to eliminate the nonspecific interactions. SPR was used successfully to follow the hybridization of the target-ssODNs with the immobilized probe-ssODNs on the platform surfaces. Complete hybridizations were achieved in 100?min. It was obtained that there was a linear relationship between relative change in delta and target concentration below 1?µm. Using imaging version of ellipsometer (IEM) allowed imaging of the surfaces and supported extra datum for the SPR results. After a very simple dehybridization protocol, MALDI-MS analysis allowed detection of the target-ssODNs hybridized on the sensor/array platforms.     

Learning and discrimination of cuticular hydrocarbons in a social insect

Social insect cuticular hydrocarbon (CHC) mixtures are among the most complex chemical cues known and are important in nest-mate, caste and species recognition. Despite our growing knowledge of the nature of these cues, we have very little insight into how social insects actually perceive and discriminate among these chemicals. In this study, we use the newly developed technique of differential olfactory conditioning to pure, custom-designed synthetic colony odours to analyse signal discrimination in Argentine ants, Linepithema humile. Our results show that tri-methyl alkanes are more easily learned than single-methyl or straight-chain alkanes. In addition, we reveal that Argentine ants can discriminate between hydrocarbons with different branching patterns and the same chain length, but not always between hydrocarbons with the same branching patterns but different chain length. Our data thus show that biochemical characteristics influence those compounds that ants can discriminate between, and which thus potentially play a role in chemical signalling and nest-mate recognition.