The overall goal of the following experiment is to detect and identify bacteria using electrochemical sensors. This is achieved by first releasing the ribosomal RNA from the bacteria to allow hybridization with the fluorinated detector probe as a second step, the detector probe bound ribosomal RNA is added to the working electrode, which allows hybridization with the capture probe on the sensor surface. Next, the anti fluorescein horse radish peroxidase conjugate is allowed to bind to the fluorinated detector probe.
Finally, in interferometric current, the intensity of which is correlated with the number of capture probe ribosomal, RNA detector probe complexes on the sensor surface can be obtained. The main advantage of this technique over culture-based methods is that bacteria can be detected and identified in about 45 minutes, avoiding the overnight weight. For a bacteria to form a visible colony on an EDGAR plate Application of this technique would improve the management of infectious diseases.
As early detection of bacterial pathogens is essential for targeted antibiotic therapy and improved clinical outcomes. Though this method is useful for diagnosis of infectious diseases that can also be applied to other diagnostic problems such as immunodiagnosis. Begin by incubating the dilated capture probe with hexane di thiol for 10 minutes at room temperature to ensure that the thiol group on the capture probe is reduced, resulting in more consistent results.
After the incubation, apply a stream of nitrogen to bear gold. 16 sensor array chips for five seconds to remove any moisture or particulates, Small volumes of liquid need to be carefully applied to the working electrode in the sensor array. Visual demonstration of this technique is critical.
As properly performing the pipetting and washing and drying techniques greatly reduces the chip to chip variability. Next, apply six microliters of the HDT dilated capture probe mix to the working electrode of all 16 sensors of the sensor array and store the sensor chips in a covered Petri dish at four degrees Celsius overnight the following day, wash the sensor chips with deionized water for two to three seconds, and then dry the chips under a stream of nitrogen for five seconds. Now apply six microliters of attri hydrochloride sodium chloride, EDTA MERCAPTOETHANOL solution to the working electrode of all 16 of the sensors, and incubate the sensors for 50 minutes in a Petri dish at room temperature.
To prepare the samples for the electrochemical sensor assay. Transfer one milliliter of bacterial culture in the log phase of growth to a micro centrifuge tube, and then centrifuge the cells for five minutes at 16, 000 gs. Please note that pathogenic bacteria should be handled in a biosafety hood.
After removing the snat, apply a pipette tip containing 10 microliters of one molar sodium hydroxide to the bottom of the micro centrifuge tube and pipette up and down a few times to thoroughly resuspend the pellet. After incubating the suspension at room temperature for five minutes, add 50 microliters of PBS containing bovine serum albumin or BSA and a fluorescein modified detector probe to neutralize the bacterial lysate. Then incubate the neutralized lysate for 10 minutes at room temperature.
Begin this step by washing the mercaptoethanol solution from one of the prepared sensor chips with deionized water for two to three seconds. After drying the chip as just demonstrated, apply four microliters of the neutralized bacteria lysate to the working electrodes. 14 of the sensors.
Then apply four microliters of the bridging oligonucleotide in the PBS plus BSA plus fluorescein modified detector probe to the two positive control sensors. Then after incubating the lysate and positive control sensors for 15 minutes, wash and then dry the entire sensor chip as before. Now apply four microliters of anti fluorescein horse radish peroxidase or HRP in PBS containing casein to the working electrodes of all 16 of the sensors.
After a 15 minute incubation, wash and dry the sensor chip. Next, apply a film well sticker to the surface of the sensor chip and load the chip into the sensor chip mount pipette 50 microliters of TMB substrate onto all 16 of the sensors and close the sensor chip mount. Finally, use a Helios chip reader to obtain pyrometry and cyclic telemetry measurements for all 16 sensors.
The just described electrochemical assay is structured similarly to a sandwich, Eliza, as shown in this figure target ribosomal, RNA hybridization with capture and detector probes is developed by a redox reaction catalyzed by HRP conjugated to anti fluorescein antibody fragments that bind the three pine fluorescein linkage on the detector probe. An important component of assay sensitivity is the surface chemistry of the gold electrode. The gold electrode is coated with mercaptoethanol and hine ol and is functionalized by the addition of dilated capture probes.
The sandwich hybridization of the target ribosomal RNA with the capture probe and the fluorescein labeled detector probe results in a complex that is detected by addition of the anti fluorescein horse radish peroxidase conjugate, and the TMB substrate. The M parametric current is generated by the redox cycle that results from the oxidation of the TMB by horseradish peroxidase and the reduction of the TMB by the electrons from the electrode. The assays demonstrated here employ a gene fluidic 16 sensor array chip.
Each of the sensors in the array contain three electrodes, working reference and auxiliary, which have contact points at the edge of the chip. The chip reader has pogo pins that connect with each of the contact points. The chip reader serves as an interface for the sensor array with a potentiometer controlled by a computer algorithm.
Here, examples of an electrochemical sensor current flow are shown. Current flow in the sensors is artificially high in the sensors during the first few seconds after parametric measurements begin. Because of the accumulation of oxidized substrate during the time from application of TMB to the sensor surface, to initiating the reader algorithm, the current flow then quickly reaches a steady state and accurate sensor readings can reliably be obtained within 60 seconds.
Sensor to sensor variability is relatively constant, so it is possible to perform 16 different sensor assays in parallel on the gene fluidic 16 sensor array chip positive and negative controls are essential as a positive control. A synthetic bridging DNA oligonucleotide that hybridizes to both the capture and detector probes and serves as a synthetic target of known concentration is used in this way. The results obtained using the bridging oligonucleotide function as an internal calibration control to minimize chip to chip variation.
The greater negative current of the electrode, the higher the signal. When testing capture and detector probes. The limit of detection should be determined by the serial dilution of a sample of known concentration as shown in this figure, there is a linear log log correlation between the sample concentration and the signal output over the dynamic range of the assay.
In these bar graphs, the representative results for samples containing esia coli and kleb seal and ammonia, which are frequently found in the urine of patients with urinary tract infection, are shown signals for most of the sensors will be low and similar, and these negative results can be pooled as the background signal reactivity with the KE probe pair distinguishes canon from e coli. The bridging oligonucleotide serves as a positive control and internal calibration standard. This technique can be applied to the detection of antibiotic resistant bacteria in clinical specimens By measuring the phenotypic response of the bacteria to antibiotics.
This technique can be utilized to determine the optimum therapy for an individual patient. After watching this video, you should have a good understanding of how to detect and identify bacteria by comparing the atric currents in the different species specific sensors in the chip.
