US 20060217893 A1
A method for detecting contaminants in a solution by determining a change in resonant frequency (ΔF) and motional resistance (ΔR) of a crystal microbalance (CM) immunosensor is presented. The method includes measuring ΔF and determining ΔR of a CM immunosenor exposed to various samples including known concentrations and a sample including an unknown concentration of the contaminant. The unknown contaminant concentration may be determined according to ΔR of the samples with the known and unknown contaminant concentrations, or ΔF of the same. If ΔR of the CM immunosensor exposed to the samples with the known contaminant concentrations more accurately reflects the known contaminant concentrations than ΔF does, the unknown contaminant concentration may be determined according to ΔR of the samples with the known contaminant concentrations and the unknown contaminant concentration. Otherwise, the unknown contaminant concentration may be determined according to ΔF of the same.
1. A method for determining an unknown contaminant concentration in a first sample, the method comprising:
determining a change in a first motional resistance of a crystal microbalance (CM) immunosensor exposed to the first sample (ΔR1);
measuring a change in a first resonant frequency of the CM immunosensor exposed to the first sample (ΔF1);
measuring a change in a second motional resistance of the CM immunosensor exposed to a plurality of second samples (ΔR2), wherein the second samples include a plurality of known contaminant concentrations;
measuring a change in a second motional frequency of the CM immunosensor exposed to the plurality of second samples (ΔF2); and
determining the unknown contaminant concentration according to ΔR2 and ΔR, or ΔF2 and ΔF1.
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This application claims priority of U.S. provisional patent application No. 60/642,335, filed Jan. 7, 2005. This provisional application is incorporated herein by reference in its entirety.
The invention was made with United States government support under Grant Number USDA/CREES 99-34211-7563 awarded by the United States Department of Agriculture. The United States government has certain rights in this invention.
Generally, quartz crystal microbalance (QCM) immunosensors for bacterial detection solely measure the resonant frequency, and the frequency shift is usually correlated to an elastic mass effect. The quartz crystal microbalance (QCM), as a simple yet powerful technique, has been widely employed in chemical and biological sensing. QCM can be designed as an immunosensor for directly detecting microorganisms without the need of labeled antibodies that are required in sandwich-type immunosensors. QCM immunosensors have been reported for rapid and specific detection of different bacteria. Most QCM immunosensors solely measure the resonant frequency (F0) using the standard oscillator technique, and the frequency change (ΔF) is usually explained by Sauerbrey equation, which states that the decrease in F0 (−ΔF) is linearly proportional to the increase in surface mass loading of QCM (Sauerbrey, 1959).
However, the Sauerbrey equation is applicable only to a thin (˜1 μm) and elastic film coupled to the crystal surface, where the mass loading can be up to 0.05% of the crystal mass. The Sauerbrey equation does not apply to the case of cells attached to the QCM surface, largely due to the softness and relatively large size of the cells. In addition to the mass effect, the changes of surface viscoelasticity and other factors also contribute to the frequency change. Due to the additive nature of these effects, the mass effect cannot be differentiated from others when only F0 is tracked.
High-frequency impedance/admittance analysis can provide more detailed information about the surface/interface changes of QCM. A QCM can be represented by a Butterworth-Van Dyke (BVD) model, which is composed of a static capacitance (C0) in parallel with a motional branch containing a motional inductance (Lm), a motional capacitance (Cm), and a motional resistance (Rm) in series. These parameters are determined by physical properties of the quartz crystal, perturbing mass layer, and contacting liquid, and can be obtained with a high-frequency impedance analyzer by fitting the measured impedance/admittance data to the BVD model. A simpler way to provide insights into the viscoelastic properties of the bound surface mass is to simultaneously monitor F0 and Rm or F0 and the dissipation factor D using a quartz crystal analyzer that is less expensive than the impedance analyzer. This method has been applied to study the behavior of adherent cells in response to chemical, biological, or physical changes in the environment.
The impedance analysis has been used to characterize a QCM immunosensor for detecting Salmonella Typhimurium. A magnetic force was utilized to collect the complexes of Salmonella-magnetic beads on the crystal surface, and Rm was found the most effective and sensitive among the four circuit parameters, which offered a detection limit of about 103 cells/ml. The sensitivity of the QCM immunosensor in the absence of magnetic beads has not been investigated nor has the measurements of Rm and F0, therefore it is unclear how much the magnetic beads could affect the detection sensitivity or which of the F0 and Rm measurements is superior in the presence or absence of the beads.
A method for detecting contaminants in a solution by determining both a change in resonant frequency (ΔF) and a change in motional resistance (ΔR) of a crystal microbalance (CM) immunosensor is presented. The method may be used to measure contaminants in samples whether or not the samples include immuno-beads, such as immuno-magnetic microbeads. The method generally includes measuring the change in frequency (ΔF) and determining change in motional resistance (ΔR) of a CM immunosenor exposed to various samples that include known concentrations of a contaminant of interest. The method further includes measuring the ΔF and determining the ΔR of a CM immunosensor exposed to a sample containing the contaminant of interest at an unknown concentration. The measurements of ΔF and ΔR create data that relate a given ΔF to a known contaminant concentration and a given ΔR to a known contaminant concentration, respectively.
The unknown contaminant concentration may be determined according to the ΔF of the samples with the known and unknown contaminant concentrations or the ΔR of the samples with the known and unknown contaminant concentrations. The unknown contaminant concentration may be determined according to the ΔR of the CM immunosensor exposed to the samples with the known contaminant concentration and the ΔR of the sample with the unknown contaminant concentration. Alternately, the unknown contaminant concentration may be determined according to ΔF of the CM immunosensor exposed to the samples with the known contaminant concentrations and the ΔF of sample with the unknown contaminant concentration. If ΔR of the CM immunosensor exposed to the samples with the known contaminant concentrations more accurately reflects the known contaminant concentrations, the unknown contaminant concentration may be determined according to the ΔR of the CM immunosensor exposed to the samples with the known contaminant concentrations and the ΔR of the sample with the unknown contaminant concentration. Otherwise, the unknown contaminant concentration may be determined according to ΔF of the CM immunosensor exposed to the samples with the known contaminant concentrations and the ΔF of sample with the unknown contaminant concentration.
A viscoelastic biosensor with enhanced sensitivity has been developed for the detection of bacterial pathogens such as Salmonella Typhimurium based on the use of immuno-magnetic microbeads and the measurement of motional resistance. For Salmonella Typhimurium, the biosensor can be fabricated using Protein A for the antibody immobilization. High-frequency impedance analysis indicates that the changes in resonant frequency and motional resistance (ΔF and ΔR) of the biosensor are significant while the changes in static capacitance, motional capacitance, and motional inductance are insignificant. ΔF and ΔR can be monitored simultaneously in real time during the biosensor fabrication and bacterial detection, and the ΔF˜ΔR diagram can be used to obtain insights into the surface characteristics. It is found that the immobilization of Protein A and antibody cause an elastic mass change while the binding of bacterial cells result in a viscoelastic change. In the direct detection of S. Typhimurium in food samples, ΔF and ΔR are proportional to the cell concentration in the range of 105 to 108, and 106 to 108 cells/ml, respectively. Using anti-Salmonella magnetic microbeads as a separator/concentrator for sample pretreatment as well as a marker for signal amplification, the detection limit is lowered to 102 cells/ml based on the ΔR measurement. There is no interference from E. coli K12 and the sample matrix.
This method is developed for food safety, and can be used in food inspection and monitoring during food processing, storage and market. With minor modification, this method can be adopted for detection of other pathogenic bacteria in food samples, including E. coli O157:H7, Listeria monocytogenes and Campylobacter jejuni. In addition to immediate applications in the food area, the method can also be used in clinical or environmental applications.
Current quartz crystal microbalance (QCM) immunosensors for bacterial detection, with few exceptions, solely measure the resonant frequency, and the frequency shift is usually correlated to an elastic mass effect. In this study, a QCM immunosensor was described for the detection of bacterial pathogens such as Salmonella Typhimurium with simultaneous measurements of the resonant frequency and motional resistance. In the case of Salmonella Typhimurium, the immunosensor was fabricated using Protein A for the antibody immobilization. High-frequency impedance analysis indicated that the changes in resonant frequency and motional resistance (ΔF and ΔR) of the QCM were significant while the changes in static capacitance, motional capacitance, and motional inductance were insignificant. ΔF and ΔR were monitored simultaneously in real time during the immunosensor fabrication and bacterial detection, and the ΔF˜ΔR diagram was used to obtain insights into the surface characteristics. It was found that the immobilization of Protein A and antibody caused an elastic mass change while the binding of bacterial cells resulted in a viscoelastic change. In the direct detection of S. Typhimurium in chicken meat sample, ΔF and ΔR were proportional to the cell concentration in the range of 105 to 108, and 106 to 108 cells/ml, respectively. Using anti-Salmonella magnetic beads as a separator/concentrator for sample pretreatment as well as a marker for signal amplification, the detection limit was lowered to 102 cells/ml based on the ΔR measurements; however, ΔF was not related to the cell concentration. No interference was observed from E. coli K12 or the sample matrix.
In this example, ΔF and the change in Rm (ΔR) of a QCM immunosensor were approximately simultaneously monitored for the detection of S. Typhimurium. In addition, the immunosensor with food samples were evaluated and the effect of immuno-magnetic beads on the detection sensitivity was investigated.
Anti-Salmonella CSA-1 antibodies (1 mg) were manufactured by Kirkegaard & Perry Laboratories (Gaithersburg, Md.). Dynabeads® anti-Salmonella (diameter 2.8 μm) were obtained from Dynal Biotech Inc. (Lake Success, N.Y.). Protein A-soluble, from S. aureus (cowan strain) cell walls, was supplied by Sigma-Aldrich (St. Louis, Mo.). Propidium iodide (PI) was purchased from Molecular Probes (Eugene, Oreg.). Phosphate buffered saline (PBS, 0.01 M, pH 7.4) containing 0.138 M NaCl and 0.0027 M KCl, and 1% (w/v) bovine serum albumin (BSA)-PBS (pH 7.4) were received from Fisher Chemical (Fair Lawn, N.J.).
Salmonella Typhimurium (ATCC 14028) as a target pathogen, and Escherichia coli K12 (ATCC 29425) as a competing bacterium were obtained from American Type Culture Collection (Rockville, Md.). The pure culture of S. Typhimurium or E. coli K12 was grown in brain heart infusion (BHI) broth (Remel, Lenexa, Kans.) at 37° C. for 20 h before use. The culture was serially diluted with physiological saline solution and the viable cell number was determined by conventional plate counting. S. Typhimurium was enumerated by surface plating on xylose lysine tergitol (XLT4) agar (Remel, Lenexa, Kans.). E. coli K12 was enumerated using sorbitol-MacConkey (SMAC) agar (Remel, Lenexa, Kans.). The undiluted cultures were heated in a 100° C. water bath for 15 min to kill all bacteria, and then diluted with PBS or sample solution to desired concentrations for further use.
Chicken breast meat purchased from a local grocery store was used as a tested sample. An amount of 25 g chicken meat was put into a Whirl-plastic bag (Nasco, Fort-Atkinson, Wis.) containing 225 ml of 0.1% buffered peptone water (Difco, Detroit, Mich.) and stomached on a Seward 400 stomacher (Seward, UK) for 2 min. The mixture was filtered by cheesecloth and then centrifuged to remove large debris and particles. An aliquot of 9 ml of the resulting meat solution was added with 1 ml of 109 cells/ml of heat-killed S. Typhimurium to make a sample solution of 108 cells/ml, which was further serially diluted to the desired concentration with the meat solution.
The inoculated sample solutions were analyzed using the QCM immunosensor directly without any other treatment or after immuno-magnetic separation (IMS). In IMS, a total of 20 μl of anti-Salmonella beads (ca. 0.1 mg or 6.6×106 beads) and 1.0 ml of sample solution containing 0-107 cells/ml of S. Typhimurium were added into micro-centrifuge tubes and vortexed for several seconds. The mixture was incubated at room temperature for 60 min with a gentle mixing. Then, the micro-centrifuge tubes were loaded into MPC-S magnetic particle concentrators (Dynal Biotech) and allowed 3 min for separating the magnetic beads from the liquid matrices. The liquid part was discarded and the resulting immuno-complexes of beads and target bacteria were resuspended in 250 μl PBS for further test with the QCM immunosensor.
The immunosensor was fabricated by immobilizing anti-Salmonella antibodies on the gold surface of AT-cut quartz crystals (International Crystal Manufacturing, Oklahoma City, Okla.), which had a diameter of 13.7 mm, a polished Au electrode (5.1 mm diameter, 1,000 Å thickness) deposited on each side, and a resonant frequency of 7.995 MHz. The crystals were pretreated with 1 M NaOH for 20 min and 1 M HCl for 5 min in sequence to obtain a clean Au surface. After each pretreatment the crystals were rinsed by spraying ethanol and water successively, and dried in a stream of nitrogen. Each of the resulting crystals was mounted on a 70-μl acrylic flow cell (International Crystal Manufacturing) as shown in
Protein A method was used for the antibody immobilization. First the crystal was flushed with 1 ml PBS to obtain a stable baseline. Secondly, the detection chamber was overflowed by 500 μl of 1 mg/ml Protein A. After 1 h incubation, the detection chamber was flushed with 1 ml PBS 5 times to rinse off the excess Protein A and to obtain a stable baseline. Thirdly, the chamber was overflowed by 500 μl of 200 μg/ml anti-Salmonella antibody solution. Also after 1 h incubation, the chamber was flushed with 1 ml PBS 5 times to rinse off the unimmobilized antibodies and to obtain a stable baseline. All the baselines were obtained in PBS at a stop-flow mode, and the differences between every two neighboring baselines were calculated as the net responses caused by the immobilization of Protein A and antibodies, respectively.
The QCM immunosensor was tested in a stop-flow mode for the detection of S. Typhimurium in PBS or chicken meat sample. First, the immunosensor was incubated with 1% BSA-PBS, blank chicken meat sample solution or 108 cells/ml of E. coli K12 solution for 1 h to block nonspecific binding sites. Then the QCM was flushed with 1 ml PBS 5 times to obtain a stable baseline. Following this, the chamber was overflowed by 1 ml (without magnetic beads) or 250 g (with magnetic beads) of sample solution. After incubation for 1 h, the chamber was flushed with 1 ml PBS 5 times to rinse off nonspecific bindings and to obtain a stable baseline. The difference between the two PBS baselines, both obtained in a stop-flow mode, was correlated to the concentration of S. Typhimurium in the sample solution.
All the experiments were conducted at room temperature, and disposable 1-ml syringes were used to push the reagent/sample solution through the detection chamber.
The QCM sensor was connected to an HP 4291A impedance analyzer (Hewlett Packard Japan, Hyogo, Japan) via an HP 16092A test fixture. The conductance and susceptance spectra (G˜f and B˜j) were measured simultaneously under a linear frequency sweep mode with 201 frequency points and a frequency span of 10 kHz covering the resonant frequency. The measured G˜f and B˜f data were fitted to the BVD model using the following equations (Tan et al., 1999),
The fluorescence images were taken on Nikon Eclipse 600 Fluorescent Microscope (Nikon Instruments, Lewisville, Tex.) using the Nikon G-2A filter set. Before fluorescent microscopy, the QCM immunosensor was incubated with 108 cells/ml of S. Typhimurium for 1 h. After being rinsed off non-specific bindings, the QCM surface was treated with 1% PI for 2 h to stain the specifically bound cells.
Although extensively used in surface/interface studies, the high-frequency impedance/admittance has been rarely applied to characterize the QCM immunosensor for bacterial detection. In this study, the QCM immunosensor was characterized step by step with the admittance analysis.
To acquire insights into the properties of the films deposited on the QCM surface, the measured admittance data were fitted to the BVD model to extract the values of the four equivalent circuit parameters C0, Lm, Rm, and Cm along with F0. Each equivalent circuit parameter has its distinct physical meaning (Martin et al., 1991; Buttry and Ward, 1992): C0 reflects the dielectric properties between the electrodes located on opposite sides of the insulating quartz crystal; Cm represents the energy stored during oscillation, which corresponds to the mechanical elasticity of the vibrating body; Lm is related to the displaced mass; and Rm is the energy dissipation during oscillation, which is closely related to viscoelasticity of the deposited films and viscosity-density of the adjacent liquid (Muramatsu et al., 1988; Lee et al., 2002). The changes of these parameters are illustrated in
Simultaneous measurements of ΔF and ΔR can differentiate an elastic mass effect from the viscosity-induced effects. ΔR is a good measure of the viscoelastic change. For an elastic mass change, ΔR will be zero and ΔF will be linearly proportional to the mass change in accordance with the Sauerbrey equation. For a QCM with only one side in contact with a Newtonian liquid, both ΔF and ΔR are linearly proportional to the squared root of the product of viscosity and density of the liquid. Hence, a pure viscosity-density change will result in a linear ΔF˜ΔR plot. As illustrated in
The ΔR˜ΔF data for the binding of Salmonella cells is displayed as line 3. At 105 cells/ml, the ΔF change was obvious but the ΔR change was negligible, indicating an elastic mass effect. When the cell concentration was higher than 106 cells/ml, both a negative ΔF shift and a positive ΔR shift were observed, and the ratio of ΔR to −ΔF was as high as 0.16˜0.48, close to or larger than the slope of the pure viscosity-density response line (line 2). Thus, the layer of bound cells was viscoelastic and the ΔF response did not obey the Sauerbrey equation. Such viscoelastic changes were also observed on certain polymeric films and cells (Zhou et al., 2000).
The QCM immunosensor was tested in a stop-flow mode for direct detection of S. Typhimurium in PBS as well as in the stomaching solution of chicken meat without using magnetic beads. Typical responses of ΔF and ΔR are given in
The calibration data for the detection of S. Typhimurium in an inoculated chicken meat sample based on the measurements of ΔF and ΔR are presented in
The relative standard deviations of the sensor-to-sensor determinations varied between 12˜29% (n=3˜6) for the ΔF measurement and 1.5˜28% for the ΔR measurement, respectively. The sample matrix did not interfere with the detection of S. Typhimurium in both the ΔF and ΔR measurements, nor did E. coli K12 although at a concentration as high as 108 cells/ml.
The QCM immunosensors for bacterial detection reported previously typically have a detection limit ranging between 105 and 107 cells/ml and a detection time of minutes to several hours. In this study, without using magnetic beads, a detection limit of 105˜10 6 cells/ml was obtained for the direct detection of S. Typhimurium. However, the infectious dosage of a foodborne pathogen such as S. Typhimurium can be as low as 15-20 cells.
In this example, the effect of magnetic beads was investigated in different ways: the magnetic force was only used for separating the Salmonella-bead complexes from sample matrix, the bead complexes were inducted to the QCM surface simply using a syringe, and ΔF and ΔR were simultaneously monitored in real time. This avoided the use of a complicated test chamber with a magnet and an ultrasonic transducer and the inconvenience of discontinuous impedance measurements.
A firm and tight attachment of bacteria causes a negative ΔF, in contrast, a flexible attachment results in a positive ΔF. In the absence of anti-Salmonella magnetic beads, the former case applied as the specific binding of Salmonella cells always led to a negative ΔF that was proportional to the cell concentration. In the presence of anti-Salmonella beads, however, ΔF was not related to the cell concentration and was either positive or negative even at the same cell concentration. This was probably because the size of the Salmonella-bead complexes was not uniform from sample to sample. S. Typhimurium is a straight rod bacterium. Typically, the width of a Salmonella cell is 0.7-1.5 μm and its length is 2-5 μm. The magnetic beads used had a diameter of 2.8 μm. The size of the Salmonella-bead complexes thus varied from several microns to tens of microns. Small complexes might generate a tight attachment and a negative ΔF, and oppositely, large complexes and aggregates could cause a flexible attachment and positive ΔF.
A significant net increase in ΔR was always observed at a cell concentration higher than 102 cells, and the net response increased with increasing cell concentration. The effect of anti-Salmonella magnetic beads can be seen more clearly in
ΔF measurement is more sensitive than the ΔR measurement in the direct detection of S. Typhimurium. When magnetic beads were used, however, the ΔR measurement was more reliable, and the sensitivity was improved by 1,000˜10,000 times. The detection limit based on the ΔR measurement was approximately 102 cells/ml, lower than those of the most reported QCM immunosensors for bacterial detection. It was also shown that simultaneous measurement of ΔF and ΔR could provide insights into the surface characteristics: the layers of immobilized Protein A and antibodies were dominantly elastic, the layer of specifically bound Salmonella cells was viscoelastic, and the magnetic beads might increase the viscoelasticity. The QCM immunosensor was successfully applied to the analysis of inoculated food samples with negligible interference form E. coli K12 and the sample matrix.
The same principle can be applied to detect other pathogenic bacteria in food, environmental and clinical samples using specific primary antibodies and immuno-magnetic beads. For example, it can be used to detect infectious bacteria in human blood and urine samples, and pathogenic bacteria in water of rivers, wells and reservoirs. It provides a rapid, sensitive, specific, inexpensive and portable biodetection method for applications in food safety and security, environmental protection and clinical diagnoses.
In addition to the microbeads, immuno-magnetic nanobeads and other types of magnetic beads can be used in this procedure for a QCM immunosensor in the detection of various pathogens. The similar detection limit, time, specificity and sensitivity are expected.
As mentioned above, QCM immunosensors, and crystal microbalance (CM) immunosensors in general, may be used in detecting contaminants in a substance. These contaminants include pathogens, bacteria, viruses, insects, arachnids and other undesirable items. An example of such a method is shown in
As shown in
ΔF of a CM immunosensor exposed to samples with the known and unknown contaminant concentrations 1102, 1104, respectively, may be measured directly. ΔR of a CM immunosensor exposed to a sample containing the known or unknown contaminant concentration 1103, 1106, respectively, may be determined by direct measurement. For example, the ΔR may be measured directly using a QCA 922. ΔR may also be measured indirectly, an example of which is shown in
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If the unknown contaminant concentration is to be determined by the ΔF of the CM immunosensor exposed to the samples with the known contaminant concentrations, the ΔF of the CM immunosensor exposed to the sample with the unknown contaminant concentration is compared with that of the known contamination concentration. The contaminant concentration corresponding to the ΔF of the known contaminant concentration that is closest to the ΔF of the unknown concentration approximately equals the unknown contaminant concentration. A similar process may be used if the unknown contaminant concentration is to be determined by the ΔR.