A recent study published in the Talanta Journal reported the development of colorimetric sensors for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ribonucleic acid (RNA)-dependent RNA polymerase (RdRp), envelope (E), and spike (S) genes.
Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) has been widely used to detect coronavirus disease 2019 (COVID-19) infection due to its high sensitivity and specificity. However, this technique needs professional interference and sophisticated equipment while also being time-consuming and expensive.
About the study
The present study showed the development of a colorimetric test using gold nanoparticles (AuNPs) for the detection of sequences coding for the SARS-CoV-2 RdRp, E, and S proteins.
Molecular beacons (MB) specific to the sequences of the E and R genes, S protein, or the D614G mutation, and AuNPs of varying sizes were generated. The size and shape of the AuNP were studied using atomic force microscopy (AFM) and transmission electron microscopy (TEM). The AuNPs were then visualized followed by sample preparation. The size and concentration of the AuNPs in the samples were determined using the ultraviolet-visible (UV-Vis) spectra.
The team functionalized citrate-stabilized AuNPs using various MB oligonucleotides after determining the number of oligonucleotides that can be conjugated to the AuNPs. Thus, different NPs contained one, two, or three MBs in their structure. The functionality of the AuNP-MB sensor was evaluated via photographs for qualitative calculations and absorbance measurements for the quantitative assessment for time periods ranging from 15 minutes to 24 hours. Samples collected from patients were amplified using reverse transcription-polymerase chain reaction (RT-PCR).
The study results showed that the oligonucleotides used for AuNP production had two different domains, among which the central one was complementary to the target regions of the SARS-CoV-2 genome. The flanking nucleotides (nt) present in this domain stabilized the oligonucleotide structure and allowed the selective recognition of target sequences in the viral genome. The sulfur and cholesterol derivatives present on each end of the oligonucleotide were required for the conjugation of the NP-oligonucleotide structures and aggregation of the NPs.
The specificity of the MBs was dependent on their sequence design and thermodynamic behavior. Among sequence design parameters, the stem length of the MB determined its kinetics and stability. Testing the functionality of 21 nanometer (nm) AuNPs with varying MB stem lengths showed that all the sensors could selectively recognize the deoxyribonucleic acid (DNA) target sequences while the stem with five nt took the least time in detecting DNA targets and was hence selected for the rest of the study.
Analyzing the impact of AuNP size on DNA detection showed that the biggest NP with a size of 38nm had higher sensitivity and lower stability as compared to the 21 nm NPs. Furthermore, 15 nm AuNPs were more stable and took longer to detect the target DNA. Finally, AuNPs between 20 and 25 nm were considered optimum for the rest of the study based on their stability and sensitivity.
The high selectivity of the AuNps was shown by the expected AuNP aggregation observed only in the presence of a specific SARS target, irrespective of the presence of other microorganisms. The efficient detection of low nm target sequences indicated high sensitivity of the AuNP-MB sensor which was further enhanced when the system combined two SARS-CoV-2-related sequences. Also, the storage time of the sensors had no effect on their functionality even when stored for three months at storage temperatures of 4°C.
Biophysical characterization of the sensor showed a reduction in absorbance in the presence of NP aggregation while clear dispersion of the NPs was observed in the absence of any target. Also, an increase in NP size was found in samples containing DNA targets. Additionally, the study showed that the interaction between the structural loop of the sensor and the target, along with the sensor-cholesterol moiety interaction accelerated the aggregation process.
The sensor had a detection limit of 7.8 x 107 to 9.6 x 107 virus copies per millilitre (mL) while SARS-CoV-2 viral loads in samples collected at five to six days post symptom onset, had 104 to 107 copies per mL, thus indicating that the sensor could accurately detect the virus in such samples. However, the study observed that the complex structure of viral RNA could potentially reduce the sensitivity of the sensor. This was negated by using modified polymerase chain reaction (PCR)-based amplification post RNA transcription. This method provided short single-stranded amplicons that allowed easy detection with sufficient sensitivity.
The study findings showed that the sensor approach modified with the amplicon production could detect the presence of SARS-CoV-2 in samples with varying viral loads. The researchers believe that this sensor could be modified to target any region on the SARS-CoV-2 genome for specific nucleic acid detection for diagnostic purposes.