Bacterial Detection & Identification Using Electrochemical Sensors

Podsumowanie

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Streszczenie

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.

Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Wprowadzenie

Using rRNA as a target molecule for bacterial detection and identification has a number of advantages. The abundance of rRNA in bacterial cells provides for a sensitivity limit as low as 250 bacteria per milliliter without the need for target amplification ¹. Bacterial rRNA contains unique species-specific sequences that are accessible to hybridization with DNA probes. Consequently, an array of electrochemical sensors can be used to identify unknown bacteria, where each sensor is functionalized with a different species-specific capture probe. Positive control sensors should be included for a synthetic oligonucleotide target that "bridges" the capture and detector probes to create an internal calibration signal.

Electrochemical sensors have a wide range of basic and translational research applications. For example, the assay described here has been used to precisely measure the effect of E. coli growth phase on rRNA and pre-rRNA copy numbers, which is of great interest to researchers interested in bacterial physiology ². The sensitivity of the electrochemical sensor assay is determined by the signal to noise ratio. A variety of signal amplification and noise reduction methods have been explored. We find that improving the chemistry of the sensor surface is key to reducing nonspecific binding of detector probe and/or HRP enzyme. In particular, a mixed monolayer of alkanedithiols and mercaptohexanol has been found to reduce background by covering the electrode surface more completely while retaining accessibility of the capture probe for target hybridization ³. These surface chemistry treatments are particularly important for assays involving complex biological samples.

Protokół

1. Functionalization of Electrochemical Sensors

1. Prepare the thiolated capture probe at a concentration of 0.05 μM in 300 μM 1,6-hexanedithiol (HDT), 10 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 1 mM EDTA and incubate in the dark at room temperature for 10 min. Incubation of the thiolated capture probe with HDT ensures that the thiol group on the capture probe is reduced, resulting in more consistent results.
2. Apply a stream of nitrogen to bare gold 16 sensor array chip(s) for 5 sec to remove moisture and/or particulates.
3. Apply 6 μl of the HDT-thiolated capture probe mix to the working electrode of all 16 sensors of the sensor array and store the sensor chip(s) in a covered Petri dish at 4 °C overnight. Thiolated capture probes bind directly to the bare gold electrode and the HDT acts to prevent overpacking of the capture probes and keep them in an extended conformation that promotes hybridization with the target.
4. The following day, wash the sensor chip with deionized H₂O for 2-3 sec and dry under a stream of nitrogen for 5 sec.
5. Apply 6 μl of 10 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 1 mM EDTA, 1 mM 6-mercapto-1-hexanol (MCH) to the working electrode of all 16 sensors and incubate for 50 min. This and all subsequent sensor chip incubations are performed in a covered Petri dish at room temperature. MCH acts as a blocking agent, filling in any gaps where the thiolated capture probe or HDT is not present on the electrode surface.

2. Sample Preparation

1. Transfer 1 ml of bacterial culture in the log phase of growth (OD₆₀₀ ≈ 0.1) to a microcentrifuge tube and centrifuge at 16,000 x g for 5 min. Remove the culture supernatant. The bacterial pellet can be processed immediately or stored at -80 °C for later use.
2. Thoroughly resuspend the bacterial pellet in 10 μl of 1 M NaOH by applying the pipette tip to the bottom of the microcentrifuge tube and pipetting up and down several times. Incubate the suspension at room temperature for 5 min.
3. Neutralize the bacterial lysate by adding 50 μl of 1 M Phosphate Buffer, pH 7.2, containing 2.5% bovine serum albumin (BSA) and 0.25 mM of a fluorescein-modified detector probe. Incubate the neutralized lysate for 10 min at room temperature. Fluorescein-modified detector probes hybridize with bacterial rRNA target molecules.

3. Electrochemical Sensor Assay

1. Wash the MCH from the sensor chip with deionized H₂O for 2-3 sec and dry under a stream of nitrogen for 5 sec.
2. Apply 4 μl of neutralized bacterial lysate to the working electrode of each of 14 sensors and incubate for 15 min. Target-detector probe complexes hybridize to immobilized thiolated capture probes.
3. Apply 4 μl of 1 nM bridging oligonucleotide in 1 M Phosphate Buffer, pH 7.2, containing 2.5% BSA and 0.25 μM fluorescein-modified detector probe to 2 positive control sensors (used for signal normalization) and incubate for 15 min.
4. Wash the sensor chip with deionized H₂O for 2-3 sec and dry under a stream of nitrogen for 5 sec.
5. Apply 4 μl of 0.5 U/ml anti-Fluorescein-HRP in 1 M Phosphate Buffer, pH 7.2, containing 0.5% casein to the working electrode of all 16 sensors and incubate for 15 min. The anti-Fluorescein-HRP binds to the immobilized fluorescein-modified detector probes.
6. Wash the sensor chip with deionized H₂O for 2-3 sec and dry under a stream of nitrogen for 5 sec.
7. Apply a film well sticker to the surface of the sensor chip and load into the sensor chip mount. Pipette 50 μl of TMB substrate onto all 16 sensors and close the sensor chip mount.
8. Obtain amperometry and cyclic voltammetry measurements for all 16 sensors using the Helios Chip Reader. Amperometric current is proportional to the rate of TMB reduction on the sensor surface (see Figure 1).

Wyniki

We describe an electrochemical assay that is structured similarly to a sandwich ELISA. As shown in Figure 1, target ribosomal RNA (rRNA) hybridization with capture and detector probes is developed by a redox reaction catalyzed by HRP conjugated to anti-fluorescein antibody fragments that bind to the 3' fluorescein linkage on the detector probe. An important component of assay sensitivity is the surface chemistry of the gold electrode. We have found that a ternary monolayer consisting of thiolated capture...

Dyskusje

The electrochemical sensor assay described here enables rapid detection of nucleic acid targets. Sensitivity and specificity depend in part on the free energy of target-probe hybridization, which in turn depends on the length and GC content of the capture and detector probes. We typically perform the hybridization steps at ambient temperature (~20 °C) ^{5, 6}. However, the hybridization steps (3.2 and 3.3) can also be performed at higher temperatures in a hybridization oven if the chip is placed in a covered ...

Materiały

Name	Company	Catalog Number	Comments
Name of the reagent	Company	Catalogue number	Comments (optional)
6-mercapto-1-hexanol (MCH)	Sigma	451088	Store at room temperature
1,6-hexanedithiol (HDT)	Sigma	H-12005	Store at room temperature
Thiolated capture probes	Operon	N/A	Store at 100 μM in 0.1x TE at -20 °C
Fluorescein-modified detector probes	Operon	N/A	Store at 100 μM in 0.1x TE at -20 °C
Bridging Oligonucleotide	Operon	N/A	Store at 100 μM in 0.1x TE at -20 °C
Anti-Fluorescein-HRP, Fab fragments	Roche	11 426 346 910	Store at 4 °C
Helios Chip Reader	GeneFluidics	GFR-2009
Sensor Chip Mount	GeneFluidics	GFR-003
Film well sticker	GeneFluidics		Shipped with sensor chips
Bare gold 16-sensor array chips	GeneFluidics	SC1000-16X-B	Store in 100% N2 at room temperature
Bovine Serum Albumin	Sigma	A7906	Store at 4 °C
1M Phosphate Buffer, pH 7.2			0.35M NaH2PO4, 0.65M K2HPO4, adjusted to pH 7.2
Blocker Casein in PBS	Pierce	37528	Dilute with an equal volume of 1M Phosphate Buffer, pH 7.2, store at 4 °C
			Table 1. Reagents and Equipment.