Microresonator mass sensors for detection of Bacillus anthracis Sterne spores in air and water

Towards the goal of developing a real-time monitoring device for microorganisms, we demonstrate the use of microcantilevers as resonant mass sensors for detection of Bacillus anthracis Sterne spores in air and liquid. The detection scheme was based on measuring resonant frequency decrease driven by thermally induced oscillations, as a result of the added mass of the spores with the use of a laser Doppler vibrometer (LDV). Viscous effects were investigated by comparing measurements in air and deionized (DI) water along with theoretical values. Moreover, biological experiments were performed which involved suspending spores onto the cantilevers and performing mass detection in air and water. For detection of spores in water, the cantilevers were functionalized with antibodies in order to fix the spores onto the surface. We demonstrate that as few as 50 spores on the cantilever can be detected in water using the thermal noise as excitation source. Measurement sensitivity of 9.23 Hz/fg for air and 0.1 Hz/fg for water were obtained. These measurements were compared with theoretical values and sources of improvement in cantilever sensitivity in a viscous medium were also discussed. It is expected that by driving the cantilevers and using higher order modes, detection of a single spore in liquids should be achievable.     

Immunoassay of prostate-specific antigen (PSA) using resonant frequency shift of piezoelectric nanomechanical microcantilever

We designed and fabricated the nanomechanical Pb(Zr0.52Ti0.48)O3 (PZT) cantilever; we demonstrated a novel electrical measurement, under a controlled ambient temperature and humidity, for label-free detection of a prostate-specific antigen (PSA); and we achieved a detection sensitivity as low as 10 pg/ml. For the fabrication of our nanomechanical PZT cantilevers, we used composite layers of Ta/Pt/PZT/Pt/SiO2 on a SiN(x) supporting layer for electrical self-sensing without external oscillators. This method allows PSA proteins to be detected via a simple electrical measurement of the resonant frequency change generated by the molecular interaction of the antigen (Ag) and the antibody (Ab). The resonant frequency shifted due to the specific binding of the PSA Ag to its Ab which is immobilized via calixcrown self-assembled monolayers on an Au surface deposited on a nanomechanical PZT cantilever. We determined the resonant frequency shift as the value of -172 Hz and -273 Hz, when the concentration of PSA Ag was 1 ng/ml, with the cantilever dimensions of 100 microm x 300 microm and 50 microm x 150 microm, respectively. Theoretical and experimental analysis suggests that the minimum detectable sensitivity for a resonant frequency shift due to a PSA Ag-Ab interaction depends on the dimensions of the nanomechanical PZT cantilever. These results also demonstrate that the experimentally measured resonant frequency shift is larger than that calculated theoretically due to the compressive stress of the PSA Ag-Ab interaction.     

Micromechanical cantilever array sensors for selective fungal immobilization and fast growth detection

We demonstrate the use of micromechanical cantilever arrays for selective immobilization and fast quantitative detection of vital fungal spores. Micro-fabricated uncoated as well as gold-coated silicon cantilevers were functionalized with concanavalin A, fibronectin or immunoglobulin G. In our experiments two major morphological fungal forms were used--the mycelial form Aspergillus niger and the unicellular yeast form Saccharomyces cerevisiae, as models to explore a new method for growth detection of eukaryotic organisms using cantilever arrays. We exploited the specific biomolecular interactions of surface grafted proteins with the molecular structures on the fungal cell surface. It was found that these proteins have different affinities and efficiencies to bind the spores. Maximum spore immobilization, germination and mycelium growth was observed on the immunoglobulin G functionalized cantilever surfaces. We show that spore immobilization and germination of the mycelial fungus A. niger and yeast S. cerevisiae led to shifts in resonance frequency within a few hours as measured by dynamically operated cantilever arrays, whereas conventional techniques would require several days. The biosensor could detect the target fungi in a range of 10(3) - 10(6) CFUml(-1). The measured shift is proportional to the mass of single fungal spores and can be used to evaluate spore contamination levels. Applications lie in the field of medical and agricultural diagnostics, food- and water-quality monitoring.     

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors detect Bacillus anthracis at 300 spores/mL

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors consisting of a piezoelectric and a borosilicate glass layer with a sensing area of 2.48 mm2 were fabricated. Antibody specific to Bacillus anthracis (BA, Sterne strain 7702) spores was immobilized on PEMC sensors, and exposed to spores (300 to 3x10(6) spores/mL). The resonant frequency decreased at a rate proportional to the spore concentration and reached a steady state frequency change of 5+/-5 Hz (n=3), 92+/-7 Hz (n=3), 500+/-10 Hz (n=3), 1030+/-10 Hz (n=2), and 2696+/-6 Hz (n=2) corresponding to 0, 3x10(2), 3x10(3), 3x10(4), and 3x10(6) spores/mL, respectively. The reduction in resonant frequency is proportional to the change in cantilever mass, and thus the observed changes are due to the attachment of spores on the sensor surface. Selectivity of the antibody-functionalized sensor was determined with samples of BA (3x10(6)/mL) mixed with Bacillus thuringiensis (BT; 1.5x10(9)/mL) in various volume ratios that yielded BA:BT ratios of 1:0, 1:125, 1:250, 1:500 and 0:1. The corresponding resonance frequency decreases were, respectively, 2345, 1980, 1310, 704 and 10 Hz. Sample containing 100% BT spores (1.5x10(9)/mL and no BA) gave a steady state frequency decrease of 10 Hz, which is within noise level of the sensor, indicating excellent selectivity. The observed binding rate constant for the pure BA and BT-containing samples ranged from 0.105 to 0.043 min-1 in the spore concentration range 300 to 3x10(6)/mL. These results show that detection of B. anthracis spore at a very low concentration (300 spores/mL) and with high selectivity in presence of another Bacillus spore (BT) can be accomplished using piezoelectric-excited millimeter-sized cantilever sensors.     

Detection and quantification of proteins using self-excited PZT-glass millimeter-sized cantilever

A composite self-excited PZT-glass cantilever (4mm in length and 2mm wide) was fabricated and used to measure the binding and unbinding of model proteins. A key feature of the cantilever is that its resonant frequency is dependent on its mass. The fabricated cantilever has mass change sensitivity in liquid of 7.2 x 10(-11)g/Hz. Resonant frequency change was measured as protein reacted or bound with the sensing glass cantilever surface. Protein concentrations, 0.1 and 1.0mg/mL, which resulted in nanogram mass change were successfully detected. The mass change sensitivity gave a total mass change of 54+/-0.45 ng for the binding of anti-rabbit IgG (biotin conjugated) to rabbit IgG immobilized cantilever and the subsequent binding of captavidin. The unbinding of anti-rabbit IgG and captavidin gave a total mass change of 54+/-1.70 ng. Fluorescence based assays showed the combined mass of both proteins in the released samples was 54+/-2.24 ng. The binding kinetics of the model proteins is modeled as first order. The initial binding rate constant of anti-rabbit IgG to rabbit IgG was 1.36+/-0.02(min(mg/mL))(-1). The initial binding rate constant of captavidin to biotinylated anti-rabbit IgG was (2.57 x 10(-1))+/-0.003(min(mg/mL))(-1). The significance of the results we report here is that millimeter-sized PZT-actuated glass cantilevers have the sensitivity to measure in real-time protein-protein binding, and the binding rate constant.     

Detection of Bacillus anthracis spores and a model protein using PEMC sensors in a flow cell at 1 mL/min

Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors of 4mm(2) sensing area were immobilized with antibody specific to Bacillus anthracis (anti-BA) spores or bovine serum albumin (anti-BSA). Detection of pathogen (Bacillus anthracis (BA) at 300 spores/mL) and BSA (1 mg/mL) were investigated under both stagnant and flow conditions. Two flow cell designs were evaluated by characterizing flow-induced resonant frequency shifts. One of the flow cells labeled SFC-2 (hold-up volume of 0.3 mL), showed small fluctuations (+/-20 Hz) around a common resonant frequency response of 217 Hz in the flow rate range of 1-17 mL/min. The total resonant frequency change obtained for the binding of 300 spores/mL in 1h was 90+/-5 Hz (n=2), and 162+/-10 Hz (n=2) under stagnant and flow conditions, respectively. Binding of antibodies, anti-BA and anti-BSA, were more rapid under flow than under stagnant conditions. The sensor was repeatedly exposed to BSA with an intermediate release step. The first and second responses to BSA were nearly identical. The total resonant frequency response to BSA was 388+/-10 (n=2) Hz under flow conditions. Kinetic analysis is carried out to quantify the effect of flow rate on antibody immobilization and the two types of detection experiments.     

Molecular Detection of <Emphasis Type="Italic">Penicillium griseofulvum</Emphasis> as the Coastal Pollution Indicator

Molecular methods were carried out to detect Penicillium griseofulvum, a dominant species related to heavy metal pollution, which was screened from marine contaminated sediments. Based on differences in internal transcribed spacer (ITS) sequences of Penicillium genus and specific isoamyl alcohol oxidase (IAO) sequences, species-specific primers AS1/RS4 and IAO1/IAO2 of Penicillium griseofulvum were designed and synthesized which were then employed in optimized PCR systems. The detection sensitivities were compared through ordinary PCR and nested-PCR using two pairs of primers, respectively. Both primer pairs could exclusively amplify destined DNA fragment from contaminated environmental samples in our researches. As for primers AS1/RS4, the detection sensitivity for spores (pure spore DNA) could be 10 fg/μl and 10 spores, respectively, and the detection sensitivity for the sediments was 10² spores/0.25 g sediments. While the detection sensitivity of IAO1/IAO2 primers was lower than that of AS1/RS4. Despite the difference in detection sensitivity, it is feasible that the species-specific primers could be used as probes for the detection of environmental pollution dominant species, Penicillium griseofulvum, since the frequency of occurrence and amount of this strain could preferably indicate the pollution degree.     

Rapid biosensor for detection of antibiotic-selective growth of Escherichia coli

A rapid biosensor for the detection of bacterial growth was developed using micromechanical oscillators coated in common nutritive layers. The change in resonance frequency as a function of the increasing mass on a cantilever array forms the basis of the detection scheme. The calculated mass sensitivity according to the mechanical properties of the cantilever sensor is approximately 50 pg/Hz; this mass corresponds to an approximate sensitivity of approximately 100 Escherichia coli cells. The sensor is able to detect active growth of E. coli cells within 1 h. The starting number of E. coli cells initially attached to the sensor cantilever was, on average, approximately 1,000 cells. Furthermore, this method allows the detection of selective growth of E. coli within only 2 h by adding antibiotics to the nutritive layers. The growth of E. coli was confirmed by scanning electron microscopy. This new sensing method for the detection of selective bacterial growth allows future applications in, e.g., rapid antibiotic susceptibility testing.     

Rapid Biosensor for Detection of Antibiotic-Selective Growth of Escherichia coli

A rapid biosensor for the detection of bacterial growth was developed using micromechanical oscillators coated in common nutritive layers. The change in resonance frequency as a function of the increasing mass on a cantilever array forms the basis of the detection scheme. The calculated mass sensitivity according to the mechanical properties of the cantilever sensor is ~50 pg/Hz; this mass corresponds to an approximate sensitivity of ~100 Escherichia coli cells. The sensor is able to detect active growth of E. coli cells within 1 h. The starting number of E. coli cells initially attached to the sensor cantilever was, on average, ~1,000 cells. Furthermore, this method allows the detection of selective growth of E. coli within only 2 h by adding antibiotics to the nutritive layers. The growth of E. coli was confirmed by scanning electron microscopy. This new sensing method for the detection of selective bacterial growth allows future applications in, e.g., rapid antibiotic susceptibility testing.     

Crocodylus niloticus (Crocodilia) is highly sensitive to water surface waves

Crocodiles show oriented responses to water surface wave stimuli but up to now behavioral thresholds are missing. This study determines the behavioral thresholds of crocodilians to water surface waves. Nile crocodiles (Crocodylus niloticus) were conditioned to respond to single-frequency water surface wave stimuli (duration 1150 ms, frequency 15, 30, 40, 60 and 80 Hz), produced by blowing air onto the water surface. Our study shows that C. niloticus is highly sensitive to capillary water surface waves. Threshold values decreased with increasing frequency and ranged between 10.3 μm (15 Hz) and 0.5 μm (80 Hz) peak-to-peak wave amplitude. For the frequencies 15 Hz and 30 Hz the sensitivity of one spectacled caiman (Caiman crocodilus) to water surface waves was also tested. Threshold values were 12.8 μm (15 Hz) down to 1.76 μm (30 Hz), i.e. close to the threshold values of C. niloticus. The surface wave sensitivity of crocodiles is similar to the surface wave sensitivity of semi-aquatic insects and fishing spiders but does not match the sensitivity of surface-feeding fishes which is higher by one to two orders of magnitude.     

Versatile, high-speed force transducer using a laser diode beam as an optical lever.

A force transducer with variable sensitivity and speed is described. Its moving element is a cantilever beam that projects vertically into a muscle bath. A brace constrains bending of the beam to a short, proximal "hinge." Rotation of the beam about the hinge is amplified 30-fold by an optical lever consisting of a laser diode beam reflected from a mirror on the cantilever to a photodiode pair. This design places the electrical components at a distance from the damp environment of the muscle bath. Large changes in sensitivity and speed can be obtained by substituting different cantilevers. Smaller changes can be made by varying the length of the hinge. A transducer with a 6-mm cantilever optimized for the study of single, skinned skeletal muscle fibers is described in detail. This device had a resonant frequency of 22 kHz and sensitivity such that the total root-mean-square noise in the circuit was more than 500-fold smaller than the expected maximum force. Variations of this device with orders of magnitude different sensitivities are also described.     

PEMC sensor's mass change sensitivity is 20 pg/Hz under liquid immersion

To enhance the mass change sensitivity of the resonating piezoelectric-excited millimeter-sized cantilever (PEMC) sensors, we reduced its length and eliminated one layer of its composite structure. As a result the mass sensitivity of the second flexural mode increased by two orders of magnitude (from 10(-9) to 10(-11)g/Hz) and the resonant frequency increased by more than 5 kHz. We demonstrate the effects of modification by detecting a model pathogen Group A Streptococcus (GAS) at 700 cells/mL. The resonant frequency change of the second mode at concentrations of 700, 7 x 10(3), 7 x 10(5), 7 x 10(6), 7 x 10(7), and 7 x 10(9)cells/mL resulted in, respectively, 3.1+/-0.5, 11.6+/-1, 15.7+/-1, 25.7+/-0.15, 28.5+/-2, and 40.5+/-3 ng (n=3 for all) of pathogen attachment. A kinetic model for the binding is proposed and verified. The observed binding rate constant was found to be in the range of 0.051-0.166 min(-1). The significance of the results we report is that the modified PEMC sensors have high mass sensitivity that pathogens can be detected at very low concentration under liquid immersion conditions.     

Nanomechanics: Opening new frontiers in bio analyses and diagnostics

Micro-fabricated silicon cantilevers arrays offer a novel label-free approach where ligand-receptor binding interactions occurring on the sensor generate nanomechanical signals like bending or a change in mass that is optically detected in-situ. We report the detection of multiple unlabelled biomolecules simultaneously down to picomolar concentrations within minutes. Differential measurements including reference cantilevers on an array of eight sensors enables sequence-specifically detection of unlabelled DNA and is suitable to detect specific gene fragments within a complete genome (gene fishing). Expression of detection of inducible genes as well as the ultimate challenge: the detection of total RNA fragments in a unspecific back ground will be shown. Ligand-receptor binding interactions, such as antigen recognition will be presented. Antibody activated cantilevers with sFv (single chain fragments) which bind to the indicator proteins show a significant improved sensitivity which is comparable with SPR (Surface Plasmon Resonance). In addition this technology offers a brought variety of receptor molecules application such as e.g. membrane protein recognition, micro-organism detection, enantiomeric separation. New coating procedures, enlargement of the active surface area by dendritic molecules as well as improvement of the receptor-cantilever chemical bond will be presented. This new findings may lead to a novel individual diagnostic assay in a combined label-free GENOMIC and PROTEOMIC biomarker sensor (COMBIOSENS).     

Micro-machined piezoelectric membrane-based immunosensor array

This paper reports a micro-machined piezoelectric membrane-based biosensor array for immunoassay. Goat immunoglobulin G (IgG) and HBsAg were immobilized as the probe molecules on the square piezoelectric membranes of the sensors that have dimensions of 3.5 microm x 500 microm x 500 microm. Due to the mass sensitive nature of these sensors, their resonant frequencies were depressed after the anti-goat IgG or anti-HBsAg was captured by the goat IgG or HBsAg. The resonant frequencies of the sensors were measured by an impedance analyzer. The experimental results demonstrate that the measured frequency change varies from 100 to 700 Hz, and the mass sensitivity of the device is estimated to be about 6.25 Hz/ng. A near linear relationship between the frequency change and the concentration of goat IgG was obtained, and the mass of the attached anti-goat IgG was calculated. The preliminary results discussed in this work indicate that the micro-machined piezoelectric membrane-based biosensor has a potential application as an immunosensor.     

Silicon photonic crystal nanocavity-coupled waveguides for error-corrected optical biosensing

A photonic crystal (PhC) waveguide based optical biosensor capable of label-free and error-corrected sensing was investigated in this study. The detection principle of the biosensor involved shifts in the resonant mode wavelength of nanocavities coupled to the silicon PhC waveguide due to changes in ambient refractive index. The optical characteristics of the nanocavity structure were predicted by FDTD theoretical methods. The device was fabricated using standard nanolithography and reactive-ion-etching techniques. Experimental results showed that the structure had a refractive index sensitivity of 10(-2) RIU. The biosensing capability of the nanocavity sensor was tested by detecting human IgG molecules. The device sensitivity was found to be 2.3±0.24×10(5) nm/M with an achievable lowest detection limit of 1.5 fg for human IgG molecules. Additionally, experimental results demonstrated that the PhC devices were specific in IgG detection and provided concentration-dependent responses consistent with Langmuir behavior. The PhC devices manifest outstanding potential as microscale label-free error-correcting sensors, and may have future utility as ultrasensitive multiplex devices.     

An analysis of local wind and air mass directions and their impact on Cladosporium distribution using HYSPLIT and circular statistics

Ecological studies that examine species-environment relationships are often limited to several meteorological parameters, i.e. mean air temperature, relative humidity, precipitation, vapour pressure deficit and solar radiation. The impact of local wind, its speed and direction are less commonly investigated in aerobiological surveys mainly due to difficulties related to the employment of specific analytical tools and interpretation of their outputs. Identification of inoculum sources of economically important plant pathogens, as well as highly allergenic bioaerosols like Cladosporium species, has not been yet explored with remote sensing data and atmospheric models such as Hybrid Single Particle Lagrangian Integrated Trajectory (HYSPLIT). We, therefore, performed an analysis of 24 h intra-diurnal cycle of Cladosporium spp. spores from an urban site in connection with both the local wind direction and overall air mass direction computed by HYSPLIT. The observational method was a volumetric air sampler of the Hirst design with 1 h time resolution and corresponding optical detection of fungal spores with light microscopy. The atmospheric modelling was done using the on-line data set from GDAS with 1° resolution and circular statistical methods. Our results showed stronger, statistically significant correlation (p ≤ 0.05) between high Cladosporium spp. spore concentration and air mass direction compared to the local wind direction. This suggested that a large fraction of the investigated fungal spores had a regional origin and must be located more than a few kilometers away from the sampling point.     

In situ, in-liquid, all-electrical detection of Salmonella typhimurium using lead titanate zirconate/gold-coated glass cantilevers at any dipping depth

Most biosensing techniques are indirect, slow, and require labeling. Even though silicon-based microcantilever sensors are sensitive and label-free, they are not suitable for in-liquid detection. More recently lead zirconate titanate (PZT) thin-film-based microcantilevers are shown to be sensitive and in situ. However, they require microfabrication and must be electrically insulated. In this study, we show that highly sensitive, in situ, Salmonella typhimurium detection can be achieved at 90% relative humidity using a lead zirconate titanate (PZT)/gold-coated glass cantilever 0.7 mm long with a non-piezoelectric 2.7 mm long gold-coated glass tip by partially dipping the gold-coated glass tip in the suspension at any depth without electrically insulating the PZT. In particular, we showed that at 90% relative humidity and with a dipping depth larger than 0.8 mm the PZT/gold-coated glass cantilever showed virtually no background resonance frequency up-shift due to water evaporation and exhibited a mass detection sensitivity of Δm/Δf = −5 × 10⁻¹¹ g/Hz. The concentration sensitivities of this PZT/gold-coated glass cantilever were 1 × 10³ and 500 cells/ml in 2 ml of liquid with a 1 and 1.5 mm dipping depth, respectively, both more than two orders of magnitude lower than the infectious dose and more than one order of magnitude lower than the detection limit of a commercial Raptor sensor.     

Missing mass effect in biosensor's QCM applications

Nowadays, liquid applications of quartz crystal microbalance (QCM) opened a way for in situ studies of proteins, vesicles and cells adsorbed from the solution onto the QCM surface. The sensitivity of QCM to the viscoelasticity of the adsorbed biomaterial can be a reason of the experimentally observed deviation from a linear dependence of QCM resonant frequency on mass deposition (the so-called Sauerbrey relation) and can limit its application for biosensoring. Presented here rigorous theoretical analysis explains the deviation from ideal mass response of soft overlayers in the contact with liquid. The fundamental result of the theory is the analog of Sauerbrey relation for layered viscous/viscoelastic medium which can be exploited for the correct physical interpretation of QCM experimental data in biofluids, in particular for measurements of the 'true' surface mass of adsorbed biomolecular films. We predict a new physical effect 'missing mass' of the sample in liquid phase measurements and compare the results given by our theory with QCM measurements on supported membranes.     

Development of quantitative real-time PCR assays for detection and quantification of surrogate biological warfare agents in building debris and leachate

Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R(2) > 0.98) over a 7-log-unit dynamic range down to 10(1) B. atrophaeus cells or spores. Quantification of S. marcescens (R(2) > 0.98) was linear over a 6-log-unit dynamic range down to 10(2) S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate.     

Gold Nanoring Arrays for Near Infrared Plasmonic Biosensing

Gold nanoring array surfaces that exhibit strong localized surface plasmon resonances (LSPR) at near infrared (NIR) wavelengths from 1.1 to 1.6 μm were used as highly sensitive real-time refractive index biosensors. Arrays of gold nanorings with tunable diameter, width, and spacing were created by the nanoscale electrodeposition of gold nanorings onto lithographically patterned nanohole array conductive surfaces over large areas (square centimeters). The bulk refractive index sensitivity of the gold nanoring arrays was determined to be up to 3,780 cm⁻¹/refractive index unit by monitoring shifts in the LSPR peak by FT-NIR transmittance spectroscopy measurements. As a first application, the surface polymerization reaction of dopamine to form polydopamine thin films on the nanoring sensor surface from aqueous solution was monitored with the real-time LSPR peak shift measurements. To demonstrate the utility of the gold nanoring arrays for LSPR biosensing, the hybridization adsorption of DNA-functionalized gold nanoparticles onto complementary DNA-functionalized gold nanoring arrays was monitored. The adsorption of DNA-modified gold nanoparticles onto nanoring arrays modified with mixed DNA monolayers that contained only 0.5 % complementary DNA was also detected; this relative surface coverage corresponds to the detection of DNA by hybridization adsorption from a 50 pM solution.