JW and MSA conceived and designed the experiments. JW, KT, and LBW performed the experiments. JW, KT, LBW, MSA, and JSK analyzed the data. JW, LBW, and JSK contributed reagents/materials/analysis tools. JW and JSK wrote the paper.
¤ Current address: Bay Area Air Quality Management District, San Francisco, California, United States of America
Tufts University has obtained a patent related to the subject of this paper, with exclusive license to CogniScent, Inc. Commercialization of the patents may result in financial benefits to the authors.
This paper demonstrates a previously unreported property of deoxyribonucleic acid—the ability of dye-labeled, solid-state DNA dried onto a surface to detect odors delivered in the vapor phase by changes in fluorescence. This property is useful for engineering systems to detect volatiles and provides a way for artificial sensors to emulate the way cross-reactive olfactory receptors respond to and encode single odorous compounds and mixtures. Recent studies show that the vertebrate olfactory receptor repertoire arises from an unusually large gene family and that the receptor types that have been tested so far show variable breadths of response. In designing biomimetic artificial noses, the challenge has been to generate a similarly large sensor repertoire that can be manufactured with exact chemical precision and reproducibility and that has the requisite combinatorial complexity to detect odors in the real world. Here we describe an approach for generating and screening large, diverse libraries of defined sensors using single-stranded, fluorescent dye–labeled DNA that has been dried onto a substrate and pulsed with brief exposures to different odors. These new solid-state DNA-based sensors are sensitive and show differential, sequence-dependent responses. Furthermore, we show that large DNA-based sensor libraries can be rapidly screened for odor response diversity using standard high-throughput microarray methods. These observations describe new properties of DNA and provide a generalized approach for producing explicitly tailored sensor arrays that can be rationally chosen for the detection of target volatiles with different chemical structures that include biologically derived odors, toxic chemicals, and explosives.
Biological systems can provide engineering guidance on how evolution has solved particular problems. In the context of detecting chemicals in either the aqueous or vapor phase, two general biological approaches have emerged. The first relies on individual highly specific single receptors (sensors), each tuned to detect a single molecular species—examples include the receptors that mediate pheromone detection in insects or those that function in neurotransmission. Specificity is achieved by narrow band design. The second approach is implemented by arrays of receptors with relatively broad responses. In this case, specificity emerges from a constellation of receptor types that recognizes the molecule of interest—the canonical example here is the olfactory receptors in the main olfactory system of vertebrates. Specificity is achieved by a “one chemical–many broadly responsive detectors” paradigm. While trying to mimic the enormous odor coding ability of biological olfaction in an “artificial nose,” we searched for molecules with the requisite combinatorial capacity to serve as odor detectors. Here we show that single-stranded DNA molecules tagged with a fluorescent reporter and deposited onto solid surfaces can respond to vapor phase odor pulses in a sequence-selective manner. These findings demonstrate new properties of nucleotide molecules that can be exploited in engineered odor detection devices. In addition, this broadband responsivity to small molecules should be explored as a functional aspect of DNA (and RNA) as they exist in the normal cellular milieu.
Short sequences of solid-state DNA can selectively signal their interactions with small molecules in the vapor phase. These observations have been implemented in odor sensing in an electronic "nose" and further suggest that in vivo responses to small molecules may represent new, nongenetic attributes of DNA.
Odor sensor arrays composed of materials that are cross-reactive have advantages over narrowly tuned receptor systems. These include combinatorial responses to sets of compounds that exceed the number of receptor or sensor types, tolerance to partial system failure, and an ability to be flexibly trained, all of which are adaptive attributes that have emerged in biological systems over evolutionary time.
In building sensors for artificial noses, there are generally two strategies that are used to develop a diversity of detectors that is reminiscent of biological receptors and thus exploits the advantages of cross-reactive arrays. First is the explicit synthesis of polymers designed to interact with defined chemical properties of a target volatile compound (for example, fluorescent conjugated polymers designed to detect nitroaromatic explosives) [
Sensors in artificial noses have not yet come close to achieving the sensor diversity and complexity found in biological olfactory systems [
In an initial test of this hypothesis, we constructed sensors of dsDNA using a standard 2.9-kb pBlueScriptSK plasmid mixed with the intercalating DNA dye YO-PRO (Molecular Probes), dried onto a polyethylene substrate material, and tested in our electronic nose device [
The odor pulse began at 0 s and lasted for 1.6 s, indicated by horizontal black bar.
(A) Responses of a sensor made from YO-PRO alone, then rinsed in 70% ethanol for 5 min.
(B) Responses of a sensor made from YO-PRO and 5 ng total pBlueScriptSK DNA. Odor dilutions, expressed as fractions of saturated vapor, were: water, 10−1; methanol (MeOH), 10−1 (∼16,700 ppm); triethylamine, 10−2 (∼750 ppm); and propionic acid, 10−1 (∼390 ppm). Each trace represents the mean of 10 presentations; error bars indicate ± 1 SD.
(A) Responses of a sensor made from OliGreen alone.
(B) Responses of a sensor made from 20 μl of 10 μM oligomer SEQ01, stained with OliGreen.
(C) Responses from SEQ01 to 10 repeated applications of 10−1 propionic acid (∼390 ppm), demonstrating return to baseline between sniffs. These 10 responses were used to calculate the mean shown in (B). See
Because different dsDNA sequences did not show odor responses that were modified by changes in sequence, we then tested sensors made from short, ssDNA oligomers stained with the fluorescent dye OliGreen (Molecular Probes). OliGreen dye alone showed a decrease in fluorescence upon exposure to propionic acid, but little change to the other odors tested (
In these tests, OliGreen and YO-PRO were applied to the DNA in solution, prior to drying onto a substrate for testing. In applying the dyes in this way, there is little control over how and where the fluorophore binds to the DNA.
To define better the dye–nucleotide interaction explicitly, we generated labeled oligonucleotides (20–24 bases long) by covalently attaching the fluorescent dye Cy3 (Amersham Biosciences) to the 5′ end during synthesis. We then adapted microarray techniques to screen these potential sensors for odor responses. An odor test chamber was constructed having the dimensions of a microscope slide to allow its use in a standard microarray scanner for examining the vapor responses from libraries of sensor sequences. Cy3-labeled oligonucleotides were spotted (∼50 μm diameter) onto cover slips, which were then mounted with the spots facing the interior volume of the test chamber (see
To measure odor responses using this method, we first tested control arrays in which the same DNA-Cy3 construct (SEQ02) was spotted at all locations. The responses of 30 replicates of the same SEQ02 construct (rows) to saturated vapors of eight odors (columns) are shown in
(A) Thirty SEQ02 control sensors (rows) tested with eight odors (columns). Pairwise Pearson correlation coefficients ranged from 0.91 to 1.00 (mean = 0.98, SD = 0.016).
(B) Twenty nine different DNA-Cy3 sensors and Cy3 alone (rows) tested with the same odor test set as (A) (columns). Pairwise correlation coefficients ranged from −0.54 to 0.98 (mean = 0.66, SD = 0.32). Dashed line denotes correlation coefficient of 0.90. Data matrices show log2 transforms of fluorescence change between clean air and odor with graded red colors indicating the degree of fluorescence increase above baseline and blue indicating the degree of decrease. Dendrograms drawn to the same scale. Abbreviations: DMMP, dimethyl methylphosphonate; DNT, dinitrotoluene.
In contrast to the correlated responses from spots having the same sequence, odor response data from 29 different DNA-Cy3 sequences (
In addition to response diversity, the sensitivities of a number of DNA-Cy3 sensors were also tested in our electronic nose device (similar to the tests shown in
(A) Responses from sequence SEQ02.
(B) Responses from a different sequence SEQ03. Sensors were made from 20 μl of 10 μM oligomer. Each data point is the mean of 10 presentations; error bars indicate ± 1 SD.
It should be noted that the responses used to plot
The solid-state, DNA-based vapor sensors described here have two key properties that are important for use in electronic noses: (1) diverse and broad odor response profiles, (
It is clear that these solid-state, DNA-based odor sensors are distinctly different from other nucleotide-based sensing materials, such as aptamers. Although aptamers have been used to detect some kinds of small, non-nucleotide ligands, these interactions have all been carried out in aqueous solution [
Analysis of sensor array and olfactory receptor properties that we have carried out using information theory [
Based on our early results published in abstract form [
Our electronic nose [
DNA was diluted to the desired concentration (0.2–50 ng/μl) in TE (10 mM Tris base, 0.5 mM EDTA, pH 8.0) or Tris-NaCl (2 mM Tris base, 11 mM NaCl, pH 8.0). Twenty μl of dilute DNA was mixed with 1 μl stock solutions of YO-PRO (1:40 in Tris-NaCl) or OliGreen (1:1 in Tris-NaCl) and incubated at room temperature for 5 min. Dye-only controls were made of 1 μl dye stock in 20 μl TE or Tris-NaCl. DNA/dye mixtures were applied to a substrate of acid-washed 16xx polyethylene silkscreen (10 mm × 12 mm) and allowed to dry for 25 min. Each sensor was rinsed in 70% ethanol for 5 min, allowed to dry, and then attached to supports on glass cover slips for testing in the electronic nose device. The dried DNA material adhered to the silkscreen mesh without occluding the openings. The exact thickness is unknown, but based on superficial appearance, it simply forms a thin film stuck to the strand supports that make up the silkscreen mesh. An air-dilution olfactometer of standard design and modeled after a system used in dog studies [
Twenty-nine ssDNA sequences labeled with Cy3 on the 5′ end were synthesized using standard phosphoramidite chemistry. The constructs were reconstituted and diluted into buffer (10 mM Tris, 50 mM NaCl, pH 8.0) at a concentration of 4 μM. Sensor constructs were then spotted onto clean 22 × 60 mm cover slips using a BioRobotics MicroGrid II.
A chamber was constructed for testing sensor array responses to odors in a Packard ScanArray 4000 microarray scanner by milling a stainless steel blank to have the outer dimensions of a standard microscope slide (1 mm thick × 25 mm wide × 75 mm long), which the scanner will accept. A rectangular hole slightly smaller (20 mm × 57 mm) than a 22 × 60 mm cover slip was cut through the center of the blank, and shoulders were cut around the hole on each side to accommodate two 22 × 60 cover slips, which, when placed on the shoulders, created an interior volume of approximately 1 cc. Three edge holes were drilled from one end of the blank into the closed volume of the chamber formed by the cover slips in order to inject odors via a 22 gauge hypodermic needle. A blank cover slip was taped into the bottom shoulder, and a sensor array cover slip was taped into the top with the DNA spots facing the interior of the chamber.
For odor testing, 30-cc glass syringes containing KimWipes saturated with different chemical compounds (or containing crystalline solid, as in the case of DNT) were used to inject odor vapor into the test chamber immediately before scanning. Ten cc of vapor was injected into each of the three edge holes and allowed to escape through the remaining holes, leading to a 30-fold exchange of chamber air. After an odor test, the chamber was opened for 15 min to allow the odor to escape. Clean humidified air was injected before each odor test to maintain constant chamber humidity. For the data analysis shown in
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The authors thank Drs. Barbara Talamo and Kathleen Dunlap for reagents; Drs. Barbara Talamo, Shana Kelley, and Andrew Chess for comments on the manuscript; and Lan Wei and John Chapin for technical assistance.
bandwidth
double-stranded DNA
single-stranded DNA