Solution-phase Detection Research Articles

Strategic Design of Non-d10 Luminescent Metal-Organic Frameworks as Dual-Mode Ultrafast and Selective Sensing Platforms for Aldehydes at the ppb Level.

Utilizing a cautious design of luminescent MOFs of non-d10 divalent transition metals based on two factors (metal nodes in an octahedral geometry to minimize nonradiative energy dissipation and tailored organic chromophores), this work reports {[Ni2(oxdz)2(tpbn)]}n (1), {[Ni2(oxdz)2(tphn)]}n (2), and {[Ni2(oxdz)2(tpon)]}n (3), synthesized at room temperature, varying the spacer length of tpbn/tphn/tpon (four, six, and eight CH2 groups, respectively). This subtle change in 1-3 is correlated to their hydrophobicity and polarizing power via water vapor sorption and selective and sensitive detection of aldehydes at the ppb level, respectively. A decrease in water vapor uptake (14.8, 8.95, and 3.19 mmol g-1 for 1-3, respectively) is observed with an increase in their hydrophobicity. On the other hand, the solution phase detection limits of acetaldehyde and benzaldehyde (2.42 and 6.71 ppb for 1, 2.77 and 4.08 ppb for 2, and 10.35 and 10.4 ppb for 3, respectively) show a similar trend for their polarizing power. The best performance of 1 is expanded to the vapor-phase detection of acetaldehyde (297% luminescence enhancement) under different pH conditions. The second mode of detection of acetaldehyde via the metal-centered electrochemical behavior of 1 provides detection limits of 38.2 and 71.5 ppb at pH 7 and 13, respectively.

Non-Equilibrium Nitrate Detection in Ionophore-Based Solid-Contact Ion-Selective Electrodes

Ion-selective electrodes (ISE’s) have benefits for solution-phase detection and quantification of ions, such as ease of operation, low cost, and high throughput compared to, e.g., ion chromatography or mass spectrometry methods. ISE devices are traditionally operated under zero-current conditions, where analyte ions passively exchange across a selective membrane barrier, yielding a Nernstian response after equilibration of Coulombic and chemical driving forces across the membrane. However, this passive mode of operation can require long equilibration times and lead to drift, for example due to electrolyte leakage and pH impacting the membrane and reference electrode. In recent years, non-equilibrium detection techniques have been of growing interest, where the ISE signal is obtained under applied electrical signal. However, the majority of this work has been devoted to cation sensing, with relatively few studies examining anion ISEs under active electrical actuation. Agricultural expansion and the corresponding uptick in application of supplemental nitrogen to soil has led to an increased demand for technologies to measure anions like nitrate – both from a production and regulation standpoint. In this work, we employ galvanostatic polarization of solid-contact ISEs as a non-equilibrium nitrate detection method. Our investigation involves coating electrodes with a redox-active charged polymer to act as a transduction layer to convert ionic to electronic signal, followed by solvent casting of a polymeric ion-selective membrane. We examine the impact of repeated rapid charging and discharging ISE electrode stacks on measurement time and stability. We compare the metrics derived from this technique to conventional open-circuit measurements using the same device. We identify benefits for this mode of operation using established nitrate membrane materials (e.g. nitrate ionophore VI membrane cocktail) that address some of the typical limitations of these conventional materials and we discuss opportunities for further refinement.

Cationic Donor–Two-Acceptor Dye–Graphene Quantum Dot Nanoconjugate for the Ratiometric Detection of Bisulfite Ions and Monitoring of SO2 Levels in Heat-Stressed Cells

When the body temperature rises above 40.6 °C, heat stress in the cells causes mitochondrial damage. This damage can lead to apoptosis, multiple organ failure, and even death. The dysregulation of SO2 levels in the mitochondria is linked to this heat stress, and its detection may act as an early indicator of heat stroke. Graphene quantum dots (GQDs), a common class of zero-dimensional carbon-based fluorescent nanomaterials, have shown immense potential as sensory probes, and it is a prospective candidate for monitoring SO2 levels in living cells. Herein, we report a combination of a donor–two acceptor (D2A) red-emissive di-picolinium salt (PPy-Br) and hydroxy-functionalized graphene quantum dots (GQDs-OH) as the fluorescence resonance energy transfer (FRET)-based ratiometric sensor for bisulfite ions detection in aqueous media. The dye-GQD nanoconjugate displays excellent photostability and good aqueous dispersibility allowing the monitoring of SO2 levels in living cells during heat stress. The PPy-Br:GQD nanoconjugate is indifferent to a large number of cations, anions, or biologically relevant species and displays a detection limit of 36 nM for the solution phase detection of bisulfite. PPy-Br is biocompatible with human cell lines and endocytoses into the cells to ensure monitoring of SO2 levels in the mitochondrial milieu. PPy-Br-treated human breast cancer cells displayed a gradual decrease in fluorescence at temperatures above 40 °C, indicating an increase in SO2 levels in heat-stressed cells. The PPy-Br:GQDs sensing conjugate is efficient in the real-time monitoring of intracellular SO2 levels and demonstrates enough prospects for further explorations as a diagnostic kit for heat stroke monitoring.

Mechanochromic and AIE active fluorescent probes for solution and vapor phase detection of picric acid: Application of logic gate

Development of organic probes through simple synthetic methodology with exceptional fluorescence properties in aqueous media, strong resistance to photobleaching, and diverse applications in solutions and vapor phase sensing are inevitably pivotal. Herein, fluorene based aggregation induced emission (AIE) active probes SM1 and SM2 were developed through a single-step Knoevenagel condensation reaction. Probes exhibited distinguished solvatochromism and reversible mechanochromism. AIE active probes SM1 and SM2 were further applied for the rapid solution-phase detection of picric acid (PA) through excellent fluorescence quenching with LOD down to 2.09 and 3.65 nM, respectively, where PET and FRET featured as vital sensing mechanisms. The sensing mechanism was supported through density functional theory (DFT) and dynamic light scattering (DLS) analyses. Probes also demonstrated the quick naked-eye colorimetric detection of PA. Dip coated fluorescent films were conveniently constructed for the efficient vapor phase and paper based detection of PA. Besides, AIE active probes were effectively employed for the quantitative recognition of PA in river and lake water samples. An INHIBIT logic device was also fabricated via the UV–visible response of probes for PA.

Detection of Explosives by SERS Platform Using Metal Nanogap Substrates.

Detecting trace amounts of explosives to ensure personal safety is important, and this is possible by using laser-based spectroscopy techniques. We performed surface-enhanced Raman scattering (SERS) using plasmonic nanogap substrates for the solution phase detection of some nitro-based compounds, taking advantage of the hot spot at the nanogap. An excitation wavelength of 785 nm with an incident power of as low as ≈0.1 mW was used to excite the nanogap substrates. Since both RDX and PETN cannot be dissolved in water, acetone was used as a solvent. TNT was dissolved in water as well as in hexane. The main SERS peaks of TNT, RDX, and PETN were clearly observed down to the order of picomolar concentration. The variations in SERS spectra observed from different explosives can be useful in distinguishing and identifying different nitro-based compounds. This result indicates that our nanogap substrates offer an effective approach for explosives identification.

Tuning Chromophore-Based LMOF Dimensionality to Enhance Detection Sensitivity for Fe3+ Ions.

Herein, we report the synthesis of two new manganese-based luminescent metal–organic frameworks (LMOFs) [Mn0.5(tipe)(1,4-ndc)]n (1) and [Mn(tipe)(1,4-ndc) (H2O)·(DMF)2·(H2O)3]n (2) [tipe = 1,1,2,2-tetrakis(4-(1H-imidazol-1-yl)phenyl)ethene (tipe) and 1,4-ndc = 1,4-naphthalenedicarboxylic acid] constructed from an aggregation-induced emission (AIE) chromophore ligand. Compound 1 can undergo a facile single-crystal-to-single-crystal transformation to form compound 2, which results in an increase in dimensionality from a two-dimensional (2D) network to a three-dimensional (3D) network. Both compounds demonstrate excellent performance for the solution-phase detection of Fe3+ ions through a significant and rapid quench in luminescence emission. Fluorescence titration experiments reveal that compound 2 is more selective toward Fe3+ compared to compound 1 because of its 3D stacking mode. The Ksv value for compound 2 (32 378 M–1) is twice as large as that for compound 1 (15 854 M–1) for the detection of Fe3+ ions. We attribute this significant increase in performance to the increase in dimensionality. In addition, compound 2 demonstrates high selectivity and sensitivity for the detection of Cr3+ cations and Cr2O72– anions.

Detection of mercury in spiked cosmetics by surface enhanced Raman spectroscopy using silver shelled iron oxide nanoparticles

Heavy metal, such as lead (Pb), arsenic (As), and mercury (Hg), contamination is a grave global issue that has affected public health via drinking water, paints, and a wide range of personal consumer products such as cosmetics. Here, we have used Surface enhanced Raman spectroscopy (SERS) for the novel solution phase detection of Hg2+ ions in spiked cosmetic (skin whitening) samples by using 2,5-Dimercapto-1,3,4-thiadiazole (DMcT) functionalized silver shelled iron oxide (Fe3O4@Ag-DMcT) nanoparticles (NPs). Fe3O4@Ag NPs (12 ± 4 nm) are the magneto-plasmonic SERS enhancers, and DMcT work as the Hg2+ reporter. We have optimized 2 mg/mL of Fe3O4@Ag:DMcT with 10−4 M of DMcT to be the best for SERS based Hg2+ detection in spiked samples of commercial skin whitening product. The samples mixed with the SERS probe were sealed in a capillary tube and placed on a magnet under the Raman spectroscope. A calibration curve of the variation of the 1360 cm−1 band of DMcT as a function of Hg2+ concentration was first determined using a cream, and a liquid phase randomly chosen cosmetics. Unknown samples spiked with low (10−8 M), and high (10−4 M) concentrations of Hg2+ could be successfully detected with ∼ 35 %, and 14.6 % error in measured intensities, respectively, with respect to the calibrated data. We estimate a limit of detection (LoD) for Hg2+ in real cosmetic sample as 1 nM (∼0.2 ppb).

Spectroelectrochemistry of Non-Aromatic Explosives in Acetonitrile and the Influence of Superoxide

The spectroelectrochemistry of non-aromatic explosive compounds, namely two nitramines (RDX and HMX), a nitrate ester (PETN), and a plastic explosive composite (Semtex 1A) in acetonitrile (AN) is reported. The redox behavior of these compounds is studied using cyclic voltammetry while monitoring changes in UV absorbance to identify transient intermediates and product species. In search of a low-cost solution-phase detection technique for explosives, we compare the spectroelectrochemistry of these compounds in the presence and absence of naturally dissolved oxygen (2 mM in AN), where the superoxide radical is co-electrogenerated during analyte reduction. Free superoxide yields a prominent UV signal in this medium (λmax = 250 nm) that the explosives’ nitro or methylene groups attenuate stoichiometrically. Overlapping UV spectra of multiple species are deconvoluted using a new strategy, spectral regional baselining, for time- and potential-resolved spectroelectrochemical (SEC) analysis. SEC data for nitrite (NO2 -), nitrate (NO3 -), and nitromethane (CH3NO2) were also collected to support proposed reaction mechanisms for the transformation of explosives that include superoxide adducts and dimerized azoxy reduced intermediates. The findings expand the understanding of high-explosive solution-phase chemistry and offer a route to novel signal transduction for (electro-)chemical sensors for energetic materials.Figure 1. Spectroelectrochemistry of PETN in aerated acetonitrile with regions denoted for differential baseline analysis. (a) 3-D projected cyclic voltammogram (black) with UV absorbance from relevant wavelength (red, unscaled) as a function of time and potential; (b) full spectra (time domain) during cyclic voltammogram. Figure 1

Electroanalysis and Spectroelectrochemistry of Nonaromatic Explosives in Acetonitrile Containing Dissolved Oxygen.

In search of a rapid, low-cost, and solution-phase detection technique for explosives, the (spectro-)electrochemistry of compounds from two major nonaromatic classes, namely nitramines (RDX and HMX) and nitrate esters (pentaerythritol tetranitrate (PETN) and the plastic explosive composite Semtex 1A) in acetonitrile (AN) is reported. In electrochemical screening, 5 μg of explosive material was detectable in 10 s by multicomponent cyclic voltammetric (CV) analysis on unmodified glassy carbon under ubiquitous environmental influences (i.e., trace water and dissolved oxygen). The explosives were identified with high recoveries under a battery of proof-of-concept testing scenarios in various matrices. In AN containing naturally dissolved oxygen (approx. 2 mM), the superoxide radical is co-electrogenerated during analyte reduction. Free superoxide yields prominent signals that the explosives attenuate quantitatively. To gain further insight into the electrochemical transformation mechanism, spectroelectrochemistry was employed to monitor changes in ultraviolet (UV) absorbance during CV and identify transient intermediates and product species, which could be targeted by future chemical sensors. Overlapping UV spectra of multiple species are deconvoluted using a new strategy, spectral regional baselining, for time- and potential-resolved spectroelectrochemical (SEC) analysis. This study shows that dissolved oxygen, hitherto an interferent purposefully removed from the solution, can be exploited advantageously in electrochemical sensing. The work expands our understanding of high-explosive solution-phase chemistry and offers a novel route to signal transduction for the sensing of energetic materials.

Spectroelectrochemistry of Explosive Aliphatic Nitro Compounds

The spectroelectrochemical behavior of non-aromatic explosive compounds, namely two nitramines (RDX and HMX), a nitrate ester (PETN), and a plastic explosive composite (Semtex 1A) in acetonitrile is reported. The electrochemical behavior of these compounds using cyclic voltammetry is performed while monitoring the UV absorbance spectra of transient and product species in situ. In search of a low-cost solution-phase detection technique, we compare the spectroelectrochemistry of these compounds in the absence and presence of naturally dissolved oxygen (~2 mM in AcN), where the superoxide radical is co-electrogenerated during explosive analyte reduction. Free superoxide yields an intense UV signal in this medium (λmax = 250 nm) that the explosives attenuate to varying degrees. We deconvolute UV spectral data through time- and potential-resolved analyses coupled with electrochemical data. It is found that superoxide interacts with the explosives through unique, previously unreported ways, likely due to strongly polarized electrostatic potentials across the poly-nitro aliphatic molecules. Reaction mechanisms will be proposed and the findings expand the understanding of high-explosive solution chemistry and offer a route to novel signal transduction for (electro-)chemical sensors for energetic materials.Figure 1. Spectroelectrochemistry of the control solution, aerated 0.1 M TBAPF6 in acetonitrile. (a) CV (ν = 50 mV s-1) in time and potential domains with the superoxide absorbance at 250 nm plotted for qualitative visualization; (b) time-resolved, full-spectrum UV data recorded during the CV. Figure 1

A stable zinc(II)-organic framework as rapid and multi-responsive luminescent sensor for metal ions in water

A very stable ZnII-organic framework, {[Zn(Titpe)0.5(bpodc)]·(H2O)3}n (1, Titpe = 1,1,2,2-tetrakis(4-(imidazol-1-yl)phenyl)ethane, bpodc = benzophenone-4,4-dicarboxylic acid), has been synthesized under hydrothermal conditions. Single-crystal X-ray analysis reveals that 1 crystallizes in the monoclinic system and P21/c space group. Its overall structure is a 3-D stacking with a porosity of 20.95% and a 4-c type topological network; it shows good stability in many solvents. Studies of the solution-phase detection of metal cations show that Fe3+ and Cr3+ ions can quench luminescent emission of 1. Fluorescence titration experiments show that 1 is more selective to Fe3+ (Ksv = 2.8 × 104 M−1) than Cr3+ (Ksv = 1.4 × 103 M−1). The solution-phase detection for anions by 1 also show high selectivity and sensitivity for Cr2O72– anions (Ksv = 1.2 × 104 M−1). With water stability and strong blue luminescence, 1 can potentially apply as a reversible multi-responsive luminescence sensor for Fe3+, Cr3+, and Cr2O72– in aqueous solutions.

An organometallic ruthenium nanocluster with conjugated aromatic ligand skeleton for explosive sensing

Abstract 9-Ethynylphenanthrene (EPT) bound to highly monodispersed Ruthenium (Ru) nanocluster (Ru:EPT) with mean diameter of \(1.5\pm 0.2\,\hbox {nm}\) and mol wt. of \({\sim }\)8600 Da was synthesized via a facile and high yield biphasic ligand exchange protocol using similar sized ethylene glycol (EG)-stabilized Ru clusters (Ru:EG) as precursor. The synthesized organometallic nanocluster was meticulously analyzed to understand its size distribution, oxidation state, crystallinity, optical and luminescence behavior and metal–ligand interfacial structure. Contrary to the extensive quenching of ligand emission by metalcore as usually observed, the ruthenium core here acts as a conductor, which conjugates surface ligands with strong emission property courtesy to an unusual vinylidene-binding motif. Thus, the synthesized nanocluster shows good luminescence property (\(\upphi = \,{\sim }\, 7\%\)) originated from the ligand skeleton and the spherical metal core restricts lateral overlap of phenanthrene moiety to cause any excimer emission. This nanocluster showed high sensitivity for solution phase detection of nitroaromatic explosives through luminescence quenching method (\(\hbox {K}_{\mathrm{SV}}\) up to \(4.98 \times 10^{4}\,\hbox {M}^{-1})\) and mimic the mechanism like conjugated organic polymer. We propose that dynamic \(\uppi -\uppi \) interaction between Ru bound phenanthrene moiety and nitroaromatic compounds followed by photoinduced electron transfer (PET), as well as Förster Resonance Energy Transfer (FRET), are the possible mechanisms behind this luminescence quenching.Graphical abstract SYNOPSIS An organometallic ruthenium nanocluster with \(\sim 8.6 \hbox { kDa mol. wt. }\) was synthesized, where aromatic phenanthrene ligands were inter-molecularly conjugated through Ru core. The Ru nanocluster showed excellent sensing performance for detection of nitroaromatic explosive molecules through luminescence quenching strategy.

Hydrogen Peroxide Coordination to Cobalt(II) Facilitated by Second-Sphere Hydrogen Bonding.

M(H2 O2 ) adducts have been postulated as intermediates in biological and industrial processes; however, only one observable M(H2 O2 ) adduct has been reported, where M is redox-inactive zinc. Herein, direct solution-phase detection of an M(H2 O2 ) adduct with a redox-active metal, cobalt(II), is described. This Co(II) (H2 O2 ) compound is made observable by incorporating second-sphere hydrogen-bonding interactions between bound H2 O2 and the supporting ligand, a trianionic trisulfonamido ligand. Thermodynamics of H2 O2 binding and decay kinetics of the Co(II) (H2 O2 ) species are described, as well as the reaction of this Co(II) (H2 O2 ) species with Group 2 cations.

Nanomaterial-based biosensors using dual transducing elements for solution phase detection.

Biosensors incorporating nanomaterials have demonstrated superior performance compared to their conventional counterparts. Most reported sensors use nanomaterials as a single transducer of signals, while biosensor designs using dual transducing elements have emerged as new approaches to further improve overall sensing performance. This review focuses on recent developments in nanomaterial-based biosensors using dual transducing elements for solution phase detection. The review begins with a brief introduction of the commonly used nanomaterial transducers suitable for designing dual element sensors, including quantum dots, metal nanoparticles, upconversion nanoparticles, graphene, graphene oxide, carbon nanotubes, and carbon nanodots. This is followed by the presentation of the four basic design principles, namely Förster Resonance Energy Transfer (FRET), Amplified Fluorescence Polarization (AFP), Bio-barcode Assay (BCA) and Chemiluminescence (CL), involving either two kinds of nanomaterials, or one nanomaterial and an organic luminescent agent (e.g. organic dyes, luminescent polymers) as dual transducers. Biomolecular and chemical analytes or biological interactions are detected by their control of the assembly and disassembly of the two transducing elements that change the distance between them, the size of the fluorophore-containing composite, or the catalytic properties of the nanomaterial transducers, among other property changes. Comparative discussions on their respective design rules and overall performances are presented afterwards. Compared with the single transducer biosensor design, such a dual-transducer configuration exhibits much enhanced flexibility and design versatility, allowing biosensors to be more specifically devised for various purposes. The review ends by highlighting some of the further development opportunities in this field.

Solution-phase detection of dual microRNA biomarkers in serum

A strategy for the simultaneous detection of multiple microRNA (miRNA) targets was developed utilizing fluorophore/quencher-labeled oligonucleotide probe sets. Two miRNA targets (miR-155 and miR-103), whose misregulation has afforded them status as putative biomarkers in certain types of cancer, were detected using our assay design. In the absence of target, the complementary fluorophore-probe and quencher-probe hybridize, resulting in a fluorescence resonance energy transfer-based quenching of the fluorescence signal. In the presence of unlabeled target, however, the antisense quencher-probe can hybridize with the target, resulting in increased fluorescence intensity as the quencher-probe is sequestered beyond the Förster radius of the fluorescent-probe. The assay design was tested in multiple matrices of buffer, cellular extract, and serum; and detection limits were found to be matrix-dependent, ranging from 0.34 to 8.89 pmol (3.4-59.3 nM) for miR-155 and 2.90-11.8 pmol (19.3-79.0 nM) for miR-103. Single, double, and triple nucleotide selectivity was also tested. Additionally, miR-155 concentrations were assessed in serum samples obtained directly from breast cancer patients without the need for RNA extraction. This assay is quantitative, possesses a low detection limit, can be applied in multiple complex matrices, and can obtain single-nucleotide selectivity. This method can be employed for the multiplex detection of solution-phase DNA or RNA targets and, more specifically, for the direct detection of serum miRNA biomarkers.

A turn-on split-luciferase sensor for the direct detection of poly(ADP-ribose) as a marker for DNA repair and cell death

Designed sensors comprising split-firefly luciferase conjugated to tandem poly(ADP-ribose) binding domains allow for the direct solution phase detection of picogram quantities of PAR and for monitoring temporal changes in poly(ADP-ribosyl)ation events in mammalian cells.

Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 × 10 −14 mol L −1. Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

TNT Detection Using Multiplexed Liquid Array Displacement Immunoassays

The presence of trace contamination of soil and groundwater with explosives is an ongoing concern, for which improved methods are required to facilitate their detection and quantification. This is true both for the monitoring of remediation and for site characterization. Immunosensors have been found effective for solution-phase detection of environmental contaminants. Our work utilized the Luminex100 (flow cytometer) to detect TNT in a multiplexed displacement immunoassay format. The Luminex100 can perform a multiplexed assay by discriminating between up to 100 different bead sets. We used this capability to evaluate four different TNT monoclonal antibodies, two recombinant TNT antibodies, and a control antibody simultaneously for the rapid detection of TNT and other nitroaromatics. TNT could be detected at 0.1 ppb and quantified over the range of 1.0 ppb to 10 ppm. In addition, the assay was shown to be effective in various matrixes such as lake water, seawater, and acetone extracts of soil. Seawater required dilution with two parts buffer to avoid loss of microspheres, while the acetone extracts were diluted 100-fold or more to minimize solvent affects.

Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization

DNA was assayed in a homogeneous format using DNA probes containing hybridization-sensitive labels. The DNA probes were prepared from complementary DNA strands in which one strand was covalently labeled on the 5′-terminus with fluorescein and the complementary strand was covalently labeled on the 3′-terminus with a quencher of fluorescein emission, either pyrenebutyrate or sulforhodamine 101. Probes prepared in this manner were able to detect unlabeled target DNA by competitive hybridization producing fluorescence signals which increased with increasing target DNA concentration. A single pair of complementary probes detected target DNA at a concentration of approximately 0.1 n m in 10 min or about 10 p m in 20–30 min. Detection of a 4 p m concentration of target DNA was demonstrated in 6 h using multiple probe pairs. The major limiting factors were background fluorescence and hybridization rates. Continuous monitoring of fluorescence during competitive hybridization allowed correction for variable sample backgrounds at probe concentration down to 20 p m; however, the time required for complete hybridization increased to greater than 1 h at probe concentrations below 0.1 n m. A promising application for this technology is the rapid detection of amplified polynucleotides. Detection of 96,000 target DNA molecules in a 50-μl sample was demonstrated following in vitro amplification using the polymerase chain reaction technique.