20 research outputs found
Protein Electrocatalysis for Direct Sensing of Circulating MicroRNAs
MicroRNAs (miRNAs) are potentially
useful biomarkers for diagnosis,
classification, and prognosis of many diseases, including cancer.
Herein, we developed a protein-facilitated electrocatalytic quadroprobe
sensor (Sens<sup>PEQ</sup>) for detection of miRNA signature of chronic
lymphocytic leukemia (CLL) in human serum. The developed signal-ON
sensor provides a compatible combination of two DNA adaptor strands
modified with four methylene blue molecules and electrocatalysis using
glucose oxidase in order to enhance the overall signal gain. This
enhanced sensitivity provided the response necessary to detect the
low-abundant serum miRNAs without preamplification. The developed
Sens<sup>PEQ</sup> is exquisitely sensitive to subtle π-stack
perturbations and capable of distinguishing single base mismatches
in the target miRNA. Furthermore, the developed sensor was employed
for profiling of three endogenous miRNAs characteristic to CLL, including
hsa-miR-16-5p, hsa-miR-21-5p, and hsa-miR-150-5p in normal healthy
serum, chronic lymphocytic leukemia Rai stage 1 (CLL-1), and stage
3 (CLL-3) sera, using a non-human cel-miR-39-3p as an internal standard.
The sensor results were verified by conventional SYBR green-based
quantitative reverse-transcription polymerase chain reaction (RT-qPCR)
analysis
Four-Way Junction Formation Promoting Ultrasensitive Electrochemical Detection of MicroRNA
MicroRNAs (miRNAs) represent a class
of biomarkers that are frequently
deregulated in cancer cells and have shown a great promise for cancer
classification and prognosis. Here, we endeavored to develop a DNA
four-way junction based electrochemical sensor (4J-SENS) for ultrasensitive
miRNA analysis. The developed sensor can be operated within the dynamic
range from 10 aM to 1 fM and detect as low as 2 aM of miR-122 (∼36
molecules per sample), without PCR amplification. Furthermore, the
4J-SENS was employed to profile endogenouse hsa-miR-122 in healthy
human and chronic lymphocyitc leukemia (CLL) patient serum, and the
results were validated by qPCR analysis
Detection of <i>C</i>. <i>parvum</i> in fruit concentrates.
<p>(A) Square wave voltammograms of the selectivity experiments performed by incubating the R4–6 aptamer-based sensor with (<i>a</i>) buffer alone, (<i>b</i>) 300 <i>Cryptosporidium parvum</i> oocysts, and (<i>c</i>) 700 <i>C</i>. <i>parvum</i> oocysts, in pineapple and mango concentrates. (B) Plot of ΔI <i>vs</i>. the tested target. All measurements were repeated three times with separate electrodes (p < 0.005).</p
Schematic representation of an electrochemical detection protocol adopted for this study.
<p>A hybrid of a thiol-modified primer and aptamer was self-assembled onto a gold nanoparticles-modified screen-printed carbon electrode (GNPs-SPCE). Binding of the <i>Cryptosporidium parvum</i> oocyst to the immobilized aptamer causes an increase in the redox current, measured by square wave voltammetry.</p
Three-Mode Electrochemical Sensing of Ultralow MicroRNA Levels
MicroRNAs (miRNAs) are an emerging class of biomarkers
that are
frequently deregulated in cancer cells and have shown great promise
for cancer classification and prognosis. In this work, we developed
a three-mode electrochemical sensor for detection and quantitation
of ultralow levels of miRNAs in a wide dynamic range of measured concentrations.
The sensor facilitates three detection modalities based on hybridization
(H-SENS), p19 protein binding (P-SENS), and protein displacement (D-SENS).
The combined three-mode sensor (HPD-SENS) identifies as low as 5 aM
or 90 molecules of miRNA per 30 μL of sample without PCR amplification,
and can be operated within the dynamic range from 10 aM to 1 μM.
The HPD sensor is made on a commercially available gold nanoparticles-modified
electrode and is suitable for analyzing multiple miRNAs on a single
electrode. This three-mode sensor exhibits high selectivity and specificity
and was used for sequential analysis of miR-32 and miR-122 on one
electrode. In addition, the H-SENS can recognize miRNAs with different
A/U and G/C content and distinguish between a fully matched miRNA
and a miRNA comprising either a terminal or a middle single base mutation.
Furthermore, the H- and P-SENS were successfully employed for direct
detection and profiling of three endogenous miRNAs, including hsa-miR-21,
hsa-miR-32, and hsa-miR-122 in human serum, and the sensor results
were validated by qPCR
Limit of detection of the aptasensor.
<p>(A) Square wave voltammograms obtained after incubating the R4–6 aptamer-based sensors with (<i>a</i>) 0, (<i>b</i>) 100, (<i>c</i>) 200, (<i>d</i>) 300, (<i>e</i>) 400, (<i>f</i>) 500, (<i>g</i>) 600, (<i>h</i>) 700, and (<i>i</i>) 800 <i>Cryptosporidium parvum</i> oocysts. (B) Calibration plot of the change in current intensity (ΔI) <i>vs</i>. number of oocysts. (C) Calibration plot of the change in potential (ΔE) <i>vs</i>. number of oocysts.</p
Electrochemical Sensing of Aptamer-Facilitated Virus Immunoshielding
Oncolytic viruses (OVs) are promising therapeutics that
selectively
replicate in and kill tumor cells. However, repetitive administration
of OVs provokes the generation of neutralizing antibodies (nAbs) that
can diminish their anticancer effects. In this work, we selected DNA
aptamers against an oncolytic virus, vesicular stomatitis virus (VSV),
to protect it from nAbs. A label-free electrochemical aptasensor was
used to evaluate the degree of protection (DoP). The aptasensor was
fabricated by self-assembling a hybrid of a thiolated ssDNA primer
and a VSV-specific aptamer. Electrochemical impedance spectroscopy
was employed to quantitate VSV in the range of 800–2200 PFU
and a detection limit of 600 PFU. The aptasensor was also utilized
for evaluating binding affinities between VSV and aptamer pools/clones.
An electrochemical displacement assay was performed in the presence
of nAbs and DoP values were calculated for several VSV-aptamer pools/clones.
A parallel flow cytometric analysis confirmed the electrochemical
results. Finally, four VSV-specific aptamer clones, ZMYK-20, ZMYK-22,
ZMYK-23, and ZMYK-28, showed the highest protective properties with
dissociation constants of 17, 8, 20, and 13 nM, respectively. Another
four sequences, ZMYK-1, -21, -25, and -29, exhibited high affinities
to VSV without protecting it from nAbs and can be further utilized
in sandwich assays. Thus, ZMYK-22, -23, and -28 have the potential
to allow efficient delivery of VSV through the bloodstream without
compromising the patient’s immune system
Anti-Fab Aptamers for Shielding Virus from Neutralizing Antibodies
Oncolytic viruses are promising therapeutics that can
selectively
replicate in and kill tumor cells. However, repetitive administration
of viruses provokes the generation of neutralizing antibodies (nAbs)
that can diminish their anticancer effect. In this work, we selected
DNA aptamers against the antigen binding fragment (Fab) of antivesicular
stomatitis virus polyclonal antibodies to shield the virus from nAbs
and enhance its in vivo survival. For the first time, we used flow
cytometry and electrochemical immunosensing to identify aptamers targeting
the Fab region of antibodies. We evaluated the aptamers in a cell-based
infection assay and found that several aptamer clones provide more
than 50% shielding of VSV from nAbs and thus have the potential to
enhance the delivery of VSV without compromising the patient’s
immune system. In addition, we developed a bifunctional label-free
electrochemical immunosensor for the quantitation of aptamer-mediated
degree of shielding and the amount of vesicular stomatitis virus (VSV)
particles. Electrochemical impedance spectroscopy was employed to
interrogate the level of VSV in a linear range from 5 × 10<sup>4</sup> to 5 × 10<sup>6</sup> PFU mL<sup>–1</sup> with
a detection limit of 10<sup>4</sup> PFU mL<sup>–1</sup>
Electrochemical Differentiation of Epitope-Specific Aptamers
DNA aptamers are promising immunoshielding agents that
could protect
oncolytic viruses (OVs) from neutralizing antibodies (nAbs) and increase
the efficiency of cancer treatment. In the present Article, we introduce
a novel technology for electrochemical differentiation of epitope-specific
aptamers (eDEA) without selecting aptamers against individual antigenic
determinants. For this purpose, we selected DNA aptamers that can
bind noncovalently to an intact oncolytic virus, vaccinia virus (VACV),
which can selectively replicate in and kill only tumor cells. The
aptamers were integrated as a recognition element into a multifunctional
electrochemical aptasensor. The developed aptasensor was used for
the linear quantification of the virus in the range of 500–3000
virus particles with a detection limit of 330 virions. Also, the aptasensor
was employed to compare the binding affinities of aptamers to VACV
and to estimate the degree of protection of VACV using the anti-L1R
neutralizing antibody in a displacement assay fashion. Three anti-VACV
aptamer clones, vac2, vac4, and vac6, showed the best immunoprotection
results and can be applied for enhanced delivery of VACV. Another
two sequences, vac5 and vac46, exhibited high affinities to VACV without
shielding it from nAb and can be further utilized in sandwich bioassays
Aptamer-Based Viability Impedimetric Sensor for Viruses
The development of aptamer-based viability impedimetric
sensor
for viruses (AptaVISens-V) is presented. Highly specific DNA aptamers
to intact vaccinia virus were selected using cell-SELEX technique
and integrated into impedimetric sensors via self-assembly onto a
gold microelectrode. Remarkably, this aptasensor is highly selective
and can successfully detect viable vaccinia virus particles (down
to 60 virions in a microliter) and distinguish them from nonviable
viruses in a label-free electrochemical assay format. It also opens
a new venue for the development of a variety of viability sensors
for detection of many microorganisms and spores