Real-time PCR for direct aptamer quantification on functionalized graphene surfaces

Santos, Viviane Cristina Fernandes dos; Almeida, Nathalie Bonatti Franco; Sousa, Thiago Alonso Stephan Lacerda de; Araújo, Eduardo Nery Duarte de; Andrade, Antero Silva Ribeiro de; Plentz, F.

doi:10.1038/s41598-019-55892-3

Cited by 11 publications

(6 citation statements)

References 37 publications

(35 reference statements)

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“…The aptamer specificity was evaluated by qPCR [ 29 – 33 ]. The ZIKV60 binding to the NS1 protein of DENV (serotypes 1, 2, 3, and 4) and YFV was also determined.…”

Section: Resultsmentioning

confidence: 99%

DNA aptamer selection and construction of an aptasensor based on graphene FETs for Zika virus NS1 protein detection

Almeida¹,

Sousa²,

Santos³

et al. 2022

Beilstein J. Nanotechnol.

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Zika virus (ZIKV) is a mosquito-borne virus that is phylogenetically close to other medically important flaviviruses with high global public health significance, such as dengue (DENV) and yellow fever (YFV) viruses. Correct diagnosis of a flavivirus infection can be challenging, particularly in world regions where more than one flavivirus co-circulates and YFV vaccination is mandatory. Acid nucleic aptamers are oligonucleotides that bind to a specific target molecule with high affinity and specificity. Because of their unique characteristics, aptamers are promising tools for biosensor development. Aptamers are usually obtained through a procedure called “systematic evolution of ligands by exponential enrichment” (SELEX). In this study, we select an aptamer (termed ZIKV60) by capillary electrophoresis SELEX (CE-SELEX) to the Zika virus non-structural protein 1 (NS1) and counterselection against the NS1 proteins of DENV (serotypes 1, 2, 3, and 4) and YFV. The ZIKV60 dissociation constant (Kd) is determined by enzyme-linked oligonucleotide assay (ELONA) and the aptamer specificity is evaluated by quantitative real-time polymerase chain reaction. ZIKV60 shows a high binding affinity to the ZIKV NS1 protein with a Kd value of 2.28 ± 0.28 nM. The aptamer presents high specificity for ZIKV NS1 compared to NS1 of DENV and YFV. Furthermore, graphene field-effect transistor devices functionalized with ZIKV60 exhibit an evident identification of NS1 protein diluted in human serum. These results point to the applicability of biosensors based on the ZIKV60 aptamer for the differential diagnosis of the Zika virus.

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“…The aptamer specificity was evaluated by qPCR [ 29 – 33 ]. The ZIKV60 binding to the NS1 protein of DENV (serotypes 1, 2, 3, and 4) and YFV was also determined.…”

Section: Resultsmentioning

confidence: 99%

DNA aptamer selection and construction of an aptasensor based on graphene FETs for Zika virus NS1 protein detection

Almeida¹,

Sousa²,

Santos³

et al. 2022

Beilstein J. Nanotechnol.

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“…A barrier to optimization of aptasensors is the inability to quantify the assay performance at each step of the protocol, as the performance is evaluated at the end by measuring the response of aptamers to the target. 5 The ability to quantify aptamers allows optimization of sensors and assays used. When developing the method with magnetic beads (see Supporting Information), we found the need to quantify the amount of aptamers that did not bind to the beads for optimization purposes.…”

Section: ■ Discussionmentioning

confidence: 99%

“…This also includes a novel way to amplify short DNA aptamers, enabling simultaneous quantification of aptamers and leptin through a single quantitative PCR (qPCR) amplification. Moreover, aptamer quantification helps to fully characterize the performance of aptasensors, which is normally evaluated at the end, making optimization challenging . The use of short aptamers is a further barrier to assay characterization, since short sequences cannot be amplified by PCR.…”

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confidence: 99%

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Aptasensor for Quantification of Leptin Through PCR Amplification of Short DNA-Aptamers

et al. 2021

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Protein quantification is traditionally performed through enzyme-linked immunosorbent assay (ELISA), which involves long preparation times. To overcome this, new approaches use aptamers as an alternative to antibodies. In this paper, we present a new approach to quantify proteins with short DNA aptamers through polymerase chain reaction (PCR) resulting in shorter protocol times with comparatively improved limit of detection. The proposed method includes a novel way to quantify both the target protein and corresponding short DNA-aptamers simultaneously, which also allows to fully characterise the performance of aptasensors. Human leptin is used as a target protein to validate this technique, because it is considered an important biomarker for obesity-related studies. In our experiments we achieved a lowest limit of detection of 100 pg/mL within less than two hours, a limit affected by the dissociation constant 1 of the leptin aptamer, which could be improved by selecting a more specific aptamer.Because of the simple and inexpensive approach, this technique can be employed for Lab-On-Chip implementations and for rapid 'on-site' quantification of proteins.

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“…However, unlike mAbs, they are developed through an in vitro process (Systematic evolution of ligands by exponential enrichment (SELEX); Ali et al., 2019), thus eliminating the need for animal hosts or immune response, and consequently enabling the development of aptamers for virtually any target (Stoltenburg et al., 2007), even complex molecular structures connected by non‐covalent bonds. Chemically modified and directly tagged aptamers, similarly to proteinaceous probes, can be used for visualization and detection of targets; however, they additionally posses properties that enable sensitive and direct quantification using real‐time quantitative polymerase chain reaction (qPCR) (dos Santos et al., 2019). Although aptamers are widely used in biomedical research, diagnostics, and environmental monitoring, they have not found their way to the field of cell wall research (DeVree et al., 2021; Ilgu and Nilsen‐Hamilton, 2016; McConnell et al., 2020; Zhou and Rossi, 2017).…”

Section: Introductionmentioning

confidence: 99%

An aptamer highly specific to cellulose enables the analysis of the association of cellulose with matrix cell wall polymers in vitro and in muro

Głazowska

Mravec

2021

The Plant Journal

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SUMMARY The current toolbox of cell wall‐directed molecular probes has been pivotal for advancing basic and application‐oriented plant carbohydrate research; however, it still exhibits limitations regarding target diversity and specificity. Scarcity of probes targeting intramolecular associations between cell wall polymers particularly hinders our understanding of the cell wall microstructure and affects the development of effective means for its efficient deconstruction for bioconversion. Here we report a detailed characterization of a cellulose‐binding DNA aptamer CELAPT MINI using a combination of various in vitro biochemical, biophysical, and molecular biology techniques. Our results show evidence for its high specificity towards long non‐substituted β‐(1‐4)‐glucan chains in both crystalline and amorphous forms. Fluorescent conjugates of CELAPT MINI are applicable as in situ cellulose probes and are well suited for various microscopy techniques, including super‐resolution imaging. Compatibility of fluorescent CELAPT MINI variants with immunodetection of cell wall matrix polymers enabled them simultaneously to resolve the fibrillar organization of complex cellulose‐enriched pulp material and to quantify the level of cellulose masking by xyloglucan and xylan. Using enzymatically, chemically, or genetically modulated Brachypodium internode sections we showed the diversity in cell wall packing among various cell types and even cell wall microdomains. We showed that xylan is the most prominent, but not the only, cellulose‐masking agent in Brachypodium internode tissues. These results collectively highlight the hitherto unexplored potential to expand the cell wall probing toolbox with highly specific and versatile in vitro generated polynucleotide probes.

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Real-time PCR for direct aptamer quantification on functionalized graphene surfaces

Cited by 11 publications

References 37 publications

DNA aptamer selection and construction of an aptasensor based on graphene FETs for Zika virus NS1 protein detection

DNA aptamer selection and construction of an aptasensor based on graphene FETs for Zika virus NS1 protein detection

Aptasensor for Quantification of Leptin Through PCR Amplification of Short DNA-Aptamers

An aptamer highly specific to cellulose enables the analysis of the association of cellulose with matrix cell wall polymers in vitro and in muro

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