2 research outputs found
Comparison of Different Strategies for the Development of Highly Sensitive Electrochemical Nucleic Acid Biosensors Using Neither Nanomaterials nor Nucleic Acid Amplification
Currently,
electrochemical nucleic acid-based biosensing methodologies
involving hybridization assays, specific recognition of RNA/DNA and
RNA/RNA duplexes, and amplification systems provide an attractive
alternative to conventional quantification strategies for the routine
determination of relevant nucleic acids at different settings. A particularly
relevant objective in the development of such nucleic acid biosensors
is the design of as many as possible affordable, quick, and simple
methods while keeping the required sensitivity. With this aim in mind,
this work reports, for the first time, a thorough comparison between
11 methodologies that involve different assay formats and labeling
strategies for targeting the same DNA. The assayed approaches use
conventional sandwich and competitive hybridization assays, direct
hybridization coupled to bioreceptors with affinity for RNA/DNA duplexes,
multienzyme labeling bioreagents, and DNA concatamers. All of them
have been implemented on the surface of magnetic beads (MBs) and involve
amperometric transduction at screen-printed carbon electrodes (SPCEs).
The influence of the formed duplex length and of the labeling strategy
have also been evaluated. Results demonstrate that these strategies
can provide very sensitive methods without the need for using nanomaterials
or polymerase chain reaction (PCR). In addition, the sensitivity can
be tailored within several orders of magnitude simply by varying the
bioassay format, hybrid length or labeling strategy. This comparative
study allowed us to conclude that the use of strategies involving
longer hybrids, the use of antibodies with specificity for RNA/DNA
heteroduplexes and labeling with bacterial antibody binding proteins
conjugated with multiple enzyme molecules, provides the best sensitivity
Toward Liquid Biopsy: Determination of the Humoral Immune Response in Cancer Patients Using HaloTag Fusion Protein-Modified Electrochemical Bioplatforms
Autoantibodies
raised against tumor-associated antigens have shown
high promise as clinical biomarkers for reliable diagnosis, prognosis,
and therapy monitoring of cancer. An electrochemical disposable biosensor
for the specific and sensitive determination of p53-specific autoantibodies
has been developed for the first time in this work. This biosensor
involves the use of magnetic microcarriers (MBs) modified with covalently
immobilized HaloTag fusion p53 protein as solid supports for the selective
capture of specific autoantibodies. After magnetic capture of the
modified MBs onto screen-printed carbon working electrodes, the amperometric
signal using the system hydroquinone/H<sub>2</sub>O<sub>2</sub> was
related to the levels of p53-autoantibodies in the sample. The biosensor
was applied for the analysis of sera from 24 patients with high-risk
of developing colorectal cancer and 6 from patients already diagnosed
with colorectal (4) and ovarian (2) cancer. The developed biosensor
was able to determine p53 autoantibodies with a sensitivity higher
than that of a commercial standard ELISA using a just-in-time produced
protein in a simpler protocol with less sample volume and easily miniaturized
and cost-effective instrumentation