Final Term Project- Summary of journal with title

“DNA Electrochemical Biosensor for the Detection of Short DNA Sequences Related to the Human
Immunodeficiency Virus”

Introduction

HIV virus is well known for its infectious properties towards human bodies which eventually may leads to AIDS
acquirement. Due to its severe impact towards human, a lot of efforts attempted to develop methods for HIV
detection. Currently several test to trace its presence has been established such as ELISA, western blot assay, or
nucleic acid hybridization using radioisotopic assays. The latter, however, is associated with hazard of
radioactive probes and problems attributed to short half-life of the material. Therefore, a new method which is
safer, faster, and cheaper must be produced.

The reported method employed an electrochemical configuration to detect hybrids obtained from assimilation of
single stranded short-DNA sequence of HIV virus with its complementary strand. Basically, the complementary
DNA strand is immobilised onto the working electrode (in this work they used carbon paste electrode)
subsequently this strand will hybridise with the single strand (SS) target DNA (21 or 42 mer single strand HIV
DNA sequence A) within the electrolyte forming a double strand nucleotides on the electrode surface. In order
to trace the hybrids, Cobalt marker (Co(phen)33+) was used. This metal will accumulate onto the hybrids surface.
To determine the amount of the accumulated marker agent, chronoamperometry technique was used. From here
they obtained the potentiogram which is useful to trace the target concentration by correlating it with calibration
curve. In this work they also included direct adsorptive chronopotentiometric stripping measurement, where the
procedure was described in their previous work, for tracing the HIV-1 related DNAs.

Procedure- discussion

Typically four major processes are involved in this work namely electrode preparation-probe immobilization,
hybridization, indicator binding to hybrid, and chronoamperometric transduction. Firstly electrode was prepared
by mixing graphite powder with mineral oil in Teflon tube, subsequently the electrode’s surface was smoothed
prior to use. Next step, is to immobilised the probe onto the electrode’s surface. Probe was immobilized due to
absorptive accumulation by applying potential (+0.5 V) into the mixture of acetate buffer solution containing
DNA probe. Second, the electrode was introduced into phosphate buffer containing 0.75 M NaCl and target
DNA for sometimes (1-30 minutes) while applying the potential at +0.5 V. Third, Cobalt indicator (Co(phen) 33+)
was introduced onto the hybrid surface by immersing the electrode into tris-HCl buffer containing 50 μM
Co(phen)33+ for 1 minute at potential of 0.5 V. Lastly, chronoamperometry was performed by supplying constant
current (-8 μA) in the Tris-HCl buffer solution.

Several single strands DNA were used for this experiment. These strands are mentioned as follow:
Target (21-mer sequence A) : 5’-ACT-GCT-AGA-GAT-TTT-CCA-CAT-3’
Immobilized probe (21-mer sequence B) : 5’-ATG-TGG-AAA-ATC-TCT-AGC-AGT-3’
Three-base mismatch (21-mer sequence A’) : 5’-ACT-GAT-AGA-CAT-TTT-CTA-CAT-3’
Target (42-mer sequence A) : 5’-TCC-CTC-AGA-CCC-TTT-TAG-TCA-GTG-TGG-
sad AAA-ATC-TCT-AGC-AGT-3’

Trace measurement was conducted by calculating the area below peaks produced in the potentiograms, These
will be referred as PSA signal. Prior to carry out measurement there are several parameters that were optimised
in this work specifically probe immobilization time, probe concentration, NaCl concentration, applied potential
during hybridization and indicator concentration. The optimum value was determined by plotting the area with
respective parameters such as time, concentration, or voltage. The optimum immobilization time was found to
be 2 minutes, this value showed that the surface was saturated by the probes. The optimum probe concentration
was found within the range of 5x10-7 up to 5x10-6. Within this range stable immobilization was obtained. The
optimum NaCl concentration was found to be 0.5 M. The applied potential during hybridization, however didn’t
seem to influence the system. Lastly the optimum indicator concentration was found to be 50 μM.
Operating conditions for assessing the sensor performances are described as follow: 0.75 M NaCl concentration,
5 minutes hybridization time, indicator concentration 5x10 -5 M, 1 minute indicator binding time, and probe
concentration of 50 μM. Two target DNAs are measured here namely 21-mer and 42-mer synthetic
oligonucletoides of HIV-1 DNA. Various concentration of target DNA were used (range of 1x10 -7-1.2x10-6), and
they observed that the linearity of the shorter oligonucleotides (linear up to 6x10-7) was greater compared to the
longer one (linear up to 3x10-7). This reduced sensitivity maybe associated with steric hindrance that influencing
the hybridization efficiency and slower rate of mass transport in the case of longer nucleotides. It was also found
that presence of polyethylene glycol could successfully accelerate the hybridization rate hence lowering
detection limit. The detection limit was found to be as low as 4 nM with signal to noise (S/N) ratio of 3.

The selectivity of the biosensor was also put into a test by introducing various different oligonucleotides during
hybridization process. The interferents were introduced as pure nucleotides or as a mixture with the target DNA.
The interefents examined in this work included ssDNA, dsDNA, scDNA, tRNA, total RNA, 36-mer DNA, and
21-mer DNA with three base mismatch compared to the actual target DNA (sequence A’). It was found that
these interferents didn’t disturb the target signal significantly whether in pure condition or within mixture. The
greatest change can be observed when the biosensor was analysing 21-mer DNA with three-base mismatch both
in pure or mixture solution.

Although it appears that the current biosensor exhibits narrow detection limit, practical HIV sensing may require
narrower detection limit. In this essence coupling the sensor with DNA amplification method like PCR is
necessary. Unfortunately this system also encountered another drawback. The electrode’s surface couldn’t be
used multiple times. Regeneration attempts only resulted in an increase of background noises. However it was
mentioned that the surface could be renewed easily and probe immobilisation could be performed in short time
(2 minutes).

In this work they also demonstrated chronopotentiometric stripping analysis for measuring the traces of HIV
related DNA. Here they tried to measure 64 μg/L of 21-mer sequence A & B of HIV-1 DNA. They found that
larger preconcentration time (accumulation time) will result in higher PSA signal. Also they noted that the
biosensor possessed higher sensitivity in sequence B detection. This is attributed to the larger existence of
Guanin in the sequence B. They managed to construct calibration curve for each DNA strands with high
correlation coefficient (0.991 and 0.995 for sequence A and B respectively). The detection limit was estimated to
be 12 and 32 μg/L for sequence B and A respectively. The relative standard deviations were found to be 11 %
and 4 % for detection of HIV-1 DNA sequences A and B with fixed concentration.

Good point of the proposed technique:

- The proposed technique offer a sensitivity of target detection while also shortening the hybridization time
effectively.
- Through this concept, necessity of using radioactive probes could be eliminated
- Relatively not a complicated method for trace level detection

Bad points of the proposed technique

- Not low enough detection limit for practical use. Therefore require coupling with DNA amplification method
to amplify samples.
- Electrode’s surface must be renewed after use leading to consumption of materials and necessity to
immobilized probes multiple times.