Nanodevices: Location of Biomarkers and Reagents within Agarose Beads of a Programmable Nano‐bio‐chip (Small 5/2011)

The slow development of cost‐effective medical microdevices with strong analytical performance characteristics is due to a lack of selective and efficient analyte capture and signaling. The recently developed programmable nano‐bio‐chip (PNBC) is a flexible detection device with analytical behavior rivaling established macroscopic methods. The PNBC system employs ≈300 μm‐diameter bead sensors composed of agarose “nanonets” that populate a microelectromechanical support structure with integrated microfluidic elements. The beads are an efficient and selective protein‐capture medium suitable for the analysis of complex fluid samples. Microscopy and computational studies probe the 3D interior of the beads. The relative contributions that the capture and detection of moieties, analyte size, and bead porosity make to signal distribution and intensity are reported. Agarose pore sizes ranging from 45 to 620 nm are examined and those near 140 nm provide optimal transport characteristics for rapid (<15 min) tests. The system exhibits efficient (99.5%) detection of bead‐bound analyte along with low (≈2%) nonspecific immobilization of the detection probe for carcinoembryonic antigen assay. Furthermore, the role analyte dimensions play in signal distribution is explored, and enhanced methods for assay building that consider the unique features of biomarker size are offered.     

Improving the Sensitivity of Fluorescence‐Based Immunoassays by Photobleaching the Autofluorescence of Magnetic Beads

In fluorescence‐based assays, usually a target molecule is captured using a probe conjugated to a capture surface, and then detected using a second fluorescently labeled probe. One of the most common capture surfaces is a magnetic bead. However, magnetic beads exhibit strong autofluorescence, which often overlaps with the emission of the reporter fluorescent dyes and limits the analytical performance of the assay. Here, several widely used magnetic beads are photobleached and their autofluorescence is reduced to 1% of the initial value. Their autofluorescence properties, including their photobleaching decay rates and autofluorescence spectra pre‐ and post‐photobleaching, and the stability of the photobleaching over a period of two months are analyzed. The photobleached beads are stable over time and their surface functionality is retained. In a high‐sensitivity LX‐200 system using photobleached magnetic beads, human interleukin‐8 is detected with a threefold improvement in detection limit and signal‐to‐noise ratio over results achievable with nonbleached beads. Since many contemporary immunoassays rely on magnetic beads as capture surfaces, prebleaching the beads may significantly improve the analytical performance of these assays. Moreover, nonmagnetic beads with low autofluorescence are also successfully photobleached, suggesting that photobleaching can be applied to various capture surfaces used in fluorescence‐based assays.     

Analytical significance of encroachment in multiplexed bead-based flow cytometric assays

Multiplexed bead-based assays, using fluorescent dye-encoded beads, are finding widespread use in various profiling studies. The need to measure multiple quantitative responses simultaneously, the development of less expensive commercial flow systems, and the ease and cost effectiveness of manufacturing bead profiling kits of varied composition have all contributed to the popularity of this assay format. Maximizing the level of multiplexing in these assays requires tight spacing of fluorescent bead populations, and this leads to some degree of overlap or "encroachment" between populations. The degree to which encroachment affects analyte signal determinations depends upon both the extent of overlap and the relative analyte signals associated with the populations. In the work reported here, the impact of encroachment upon analyte signal for a subset of beads belonging to a multiplexed cytokine assay has been modeled and empirically evaluated.     

Resistive pulse sensing of analyte-induced multicomponent rod aggregation using tunable pores

Resistive pulse sensing is used to monitor individual and aggregated rod-shaped nanoparticles as they move through tunable pores in elastomeric membranes. By comparing particles of similar dimensions, it is demonstrated that the resistive pulse signal of a rod is fundamentally different from that of a sphere. Rods can be distinguished using two measurements: the blockade event magnitude (Δi(p) ), which reveals the particle's size, and the full width at half maximum (FWHM) duration, which relates to the particle's speed and length. While the observed Δi(p) values agree well with simulations, the measured FWHM times are much larger than expected. This increase in dwell time, caused by rods moving through the pore in various orientations, is not observed for spherical particles. These differences are exploited in a new agglutination assay using rod-shaped particles. By controlling the surface chemistry and location of the capture ligand, rods are made to form either long "end-on-end" or wide "side-on" aggregates upon the addition of an analyte. This observation will facilitate multiplexed detection in agglutination assays, as particles with a particular aspect ratio can be distinguished by two measurements. This is first demonstrated with a biotinylated target and avidin capture probe, followed by the detection of platelet-derived growth factor (PDGF-BB) using an aptamer capture probe, with limits of detection down to femtomolar levels.     

Design of Hierarchical Beads for Efficient Label‐Free Cell Capture

Defined hierarchical materials promise cell analysis and call for application‐driven design in practical use. The further issue is to develop advanced materials and devices for efficient label‐free cell capture with minimum instrumentation. Herein, the design of hierarchical beads is reported for efficient label‐free cell capture. Silica nanoparticles (size of ≈15 nm) are coated onto silica spheres (size of ≈200 nm) to achieve nanoscale surface roughness, and then the rough silica spheres are combined with microbeads (≈150–1000 µm in diameter) to assemble hierarchical structures. These hierarchical beads are built via electrostatic interaction, covalent bonding, and nanoparticle adherence. Further, after functionalization by hyaluronic acid (HA), the hierarchical beads display desirable surface hydrophilicity, biocompatibility, and chemical/structural stability. Due to the controlled surface topology and chemistry, HA‐functionalized hierarchical beads afford high cell capture efficiency up to 98.7% in a facile label‐free manner. This work guides the development of label‐free cell capture techniques and contributes to the construction of smart interfaces in bio‐systems.     

DNA-wrapped carbon nanotubes as sensitive electrochemical labels in controlled-assembly-mediated signal transduction for the detection of sequence-specific DNA

A novel electrochemical strategy that uses DNA-wrapped carbon nanotubes (CNTs) as electrochemical labels is developed for sensitive and selective detection of sequence-specific DNA. The presence of target DNA mediates the formation of a sandwiched complex between the DNA-wrapped CNT and a hairpin DNA capture probe immobilized on magnetic beads. This allows target-selective collection of the CNT labels by magnetic separation and transfer on the electrode surface modified with an insulating self-assembled monolayer (SAM). After treatment with N,N-dimethylformamide, the collected sandwiched complex releases the bare CNTs and facilitates the removal of magnetic beads from the electrode surface. The bare CNTs can then assemble on the SAM-modified electrode surface and mediate efficient electron transfer between the electrode and the electroactive species in the solution with a strong current signal generated. The results indicate that the developed strategy shows a sensitive response to target DNA with a desirable signal gain and a low detection limit of 0.9 pM. This strategy is also demonstrated to provide excellent differentiation of single-base mismatch in target DNA. It is expected that this electrochemical strategy may hold great potential as a novel platform for clinical diagnostics and genetic analysis.     

Modeling analyte transport and capture in porous bead sensors

Porous agarose microbeads, with high surface to volume ratios and high binding densities, are attracting attention as highly sensitive, affordable sensor elements for a variety of high performance bioassays. While such polymer microspheres have been extensively studied and reported on previously and are now moving into real-world clinical practice, very little work has been completed to date to model the convection, diffusion, and binding kinetics of soluble reagents captured within such fibrous networks. Here, we report the development of a three-dimensional computational model and provide the initial evidence for its agreement with experimental outcomes derived from the capture and detection of representative protein and genetic biomolecules in 290 μm porous beads. We compare this model to antibody-mediated capture of C-reactive protein and bovine serum albumin, along with hybridization of oligonucleotide sequences to DNA probes. These results suggest that, due to the porous interior of the agarose bead, internal analyte transport is both diffusion and convection based, and regardless of the nature of analyte, the bead interiors reveal an interesting trickle of convection-driven internal flow. On the basis of this model, the internal to external flow rate ratio is found to be in the range of 1:170 to 1:3100 for beads with agarose concentration ranging from 0.5% to 8% for the sensor ensembles here studied. Further, both model and experimental evidence suggest that binding kinetics strongly affect analyte distribution of captured reagents within the beads. These findings reveal that high association constants create a steep moving boundary in which unbound analytes are held back at the periphery of the bead sensor. Low association constants create a more shallow moving boundary in which unbound analytes diffuse further into the bead before binding. These models agree with experimental evidence and thus serve as a new tool set for the study of bioagent transport processes within a new class of medical microdevices.     

Microfabricated renewable beads-trapping/releasing flow cell for rapid antigen-antibody reaction in chemiluminescent immunoassay

A renewable flow cell integrating a microstructured pillar-array filter and a pneumatic microvalve was microfabricated to trap and release beads. A bead-based immunoassay using this device was also developed. This microfabricated device consists of a microfluidic channel connecting to a beads chamber in which the pillar-array filter is built. Underneath the filter, there is a pneumatic microvalve built across the chamber. Such a device can trap and release beads in the chamber by "closing" or "opening" the microvalve. On the basis of the pneumatic microvalve, the device can trap beads in the chamber before performing an assay and release the used beads after the assay. Therefore, this microfabricated device is suitable for "renewable surface analysis". A model analyte, 3,5,6-trichloropyridinol (TCP), was chosen to demonstrate the analytical performance of the device. The entire fluidic assay process, including beads trapping, immuno binding, beads washing, beads releasing, and chemiluminesence signal collection, could be completed in 10 min. The immunoassay of TCP using this microfabricated device showed a linear range of 0.20-70 ng/mL with a limit of detection of 0.080 ng/mL. The device was successfully used to detect TCP spiked in human plasma at the concentration range of 1.0-50 ng/mL, with an analytical recovery of 81-110%. The results demonstrated that this device can provide a rapid, sensitive, reusable, low-cost, and automatic tool for detecting various biomarkers in biological fluids.     

SERS biodetection using gold-silica nanoshells and nitrocellulose membranes

We have developed a rapid, reproducible, easy to execute, surface enhanced Raman scattering (SERS) method for detection of low volumes and total amounts of biological antigens using an analyte capture system derived from methods commonly used in Western blotting. Our method is a "half-sandwich" assay with an antigen detection scheme that employs a nitrocellulose (NC) membrane with 200 nm pore size to capture subnanograms of analyte and concentrate them in a small area from applied volumes as low as one microliter. The SERS probes used for detection consist of gold-silica nanoshells modified with a two-component mixed monolayer system. One component consists of a poly(ethylene glycol) (PEG)-modified Raman-active chromophore bound to the gold surface which allows for SERS detection and imparts particle stability. The second component uses (ortho-pyridyl) disulfide-PEG-succinimidyl ester to couple the recognition antibody to the particle surface. By controlling the reaction time and concentration of thiols, a mixed monolayer is prepared on the nanoshell surface with the ability to recognize low concentrations of analyte and generate reproducible SERS signals. Using this strategy, we have achieved SERS signals that are proportional to antigen present on the membrane allowing detection of total antigen amounts as low as 1.25 ng for some cases. The performance of this new SERS bioassay has been tested with a variety of potential antigens, demonstrating the potential for multiplexed detection of analytes.     

Equilibration-based preconcentrating minicolumn sensors for trace level monitoring of radionuclides and metal ions in water without consumable reagents

A sensor technique is described that captures analyte species on a preconcentrating minicolumn containing a selective solid-phase sorbent. In this approach, the sample is pumped through the column until the sorbent phase is fully equilibrated with the sample concentration, and the exit concentration equals the inlet concentration. On-column detection of the captured analytes using radiometric and spectroscopic methods is demonstrated. In trace level detection applications, this sensor provides a steady-state signal that is proportional to sample analyte concentration and is reversible. The method is demonstrated for the detection of Tc-99 using anion-exchange beads mixed with scintillating beads and light detection, Sr-90 using SuperLig 620 beads mixed with scintillating beads and light detection; and hexavalent chromium detection using anion-exchange beads with spectroscopic detection. Theory has been developed to describe the signal at equilibration and to describe analyte uptake as a function of volume and concentration, using parameters and concepts from frontal chromatography. It is shown that experimental sensor behavior closely matches theoretical predictions and that effective sensors can be prepared using low plate number columns. This sensor modality has many desirable characteristics for in situ sensors for trace level contaminant long-term monitoring where the use of consumable reagents for sensor regeneration would be undesirable. Initial experiments in groundwater matrixes demonstrated the detection of Tc-99 at drinking water level standards (activity of 0.033 Bq/mL) and detection of hexavalent chromium to levels below drinking water standards of 50 ppb.     

消失模EPS模型材料表征及性能的研究

为了探究消失模EPS模型的表面粗糙度影响因素、应力应变特征以及消失模EPS模型材料的热解规律和内部胞腔结构形态,达到降低消失模铸件的表面粗糙度,科学制定消失模工艺过程的控制参数,选择适合消失模工艺的合金材料,控制铸件内碳化物残留量,提高消失模铸件的表面质量和力学性能.本文采用激光粒度分析仪对用于消失模EPS产品模型成形...     

Green fluorescent protein in the design of a living biosensing system for L-arabinose

Analysis of monosaccharides is typically performed using analytical systems that involve a separation step followed by a detection step. The separation step is usually necessary because of the high degree of structural similarity between different monosaccharides. A novel sensing system for monosaccharides is described here in which living bacteria were designed to detect a model monosaccharide, L-arabinose, without the need for a separation step. In such sensing systems, analytes are detected by employing the selective recognition properties found in certain bacterial proteins. These systems are designed so that a reporter protein is expressed by the bacteria in response to the analyte. The concentration of the analyte can be related to the signal generated by the reporter protein. In the sensing system described here, the green fluorescent protein (GFP) was used as the reporter protein. L-Arabinose concentrations can be determined by monitoring the fluorescence emitted by the bacteria at 509 nm after excitation of GFP at 395 nm. The system can detect L-arabinose at concentrations as low as 5 x 10(-7) M and is selective over D-arabinose, the stereoisomer of the analyte, as well as over a variety of pentose and hexose sugars.     

Universal approach for selective trace metal determinations via sequential injection-bead injection-lab-on-valve using renewable hydrophobic bead surfaces as reagent carriers

A new concept is presented for selective and sensitive determination of trace metals via electrothermal atomic absorption spectrometry based on the principle of bead injection (BI) with renewable reversed-phase surfaces in a sequential injection-lab-on-valve (SI-LOV) mode. The methodology involves the use of poly(styrene-divinylbenzene) beads containing pendant octadecyl moieties (C18-PS/DVB), which are preimpregnated with a selective organic metal chelating agent prior to the automatic manipulation of the beads in the microbore conduits of the LOV unit. By adapting this approach, the immobilization of the most suitable chelating agent can be effected irrespective of the kinetics involved, optimal reaction conditions can be used for implementing the chelating reaction of the target metal analyte with the immobilized re-agent, and an added degree of freedom is offered in selecting the most favorable elution mode in order to attain the highest sensitivity. The potential of the SI-BI-LOV scheme is demonstrated by taking Cr(VI) as a model analyte, using a 1,5-diphenylcarbazide (DPC)-loaded bead column as the active microzone. As this reaction requires the use of high acidity, it is also shown that the bead material exhibits excellent chemical stability at low pH values. On-line pH sample adjustment prevents alteration of the original distribution of chromium species while ensuring fast rates for the DPC-Cr(VI) reaction. The proposed procedure was successfully applied to the determination of trace levels of Cr(VI) in natural waters containing high levels of dissolved salts (such as seawater and hard tap water) without requiring any dilution step. Method validation was performed by determination of total chromium in an NIST standard reference material (NIST 1640, natural water) after Cr(III) oxidation, and the results were in good agreement with the certified value.     

DNA-based hybridization chain reaction for amplified bioelectronic signal and ultrasensitive detection of proteins

This work reports a novel electrochemical immunoassay protocol with signal amplification for determination of proteins (human IgG here used as a model target analyte) at an ultralow concentration using DNA-based hybridization chain reaction (HCR). The immuno-HCR assay consists of magnetic immunosensing probes, nanogold-labeled signal probes conjugated with the DNA initiator strands, and two different hairpin DNA molecules. The signal is amplified by the labeled ferrocene on the hairpin probes. In the presence of target IgG, the sandwiched immunocomplex can be formed between the immobilized antibodies on the magnetic beads and the signal antibodies on the gold nanoparticles. The carried DNA initiator strands open the hairpin DNA structures in sequence and propagate a chain reaction of hybridization events between two alternating hairpins to form a nicked double-helix. Numerous ferrocene molecules are formed on the neighboring probe, each of which produces an electrochemical signal within the applied potentials. Under optimal conditions, the immuno-HCR assay presents good electrochemical responses for determination of target IgG at a concentration as low as 0.1 fg mL(-1). Importantly, the methodology can be further extended to the detection of other proteins or biomarkers.     

Trace-concentration detection of cobalt in a liquid flow cell by degenerate four-wave mixing using low-power off-resonant laser excitation.

Optical phase conjugation by degenerate four-wave mixing (D4WM) in an absorbing metal-ion solution using a low-power argon-ion laser as the excitation source is demonstrated. This nonlinear laser technique can be used as a sensitive analytical spectroscopic method for trace-concentration measurement of metal ions in a small-volume continuously flowing analyte cell. Several important characteristics are discussed, including the effects of solvent properties, excitation wave-length, laser intensity, and analyte absorptivity on signal intensity. Detection of 0.26 ng (4.4 pmol) of cobalt inside the laser probe volume of 0.14 microL is reported using an excitation wavelength that is 136 nm away from the maximum absorption wavelength of the analyte solution. The minimum absorbance measured in our D4WM experiment is 2.0 X 10(-5) without complex formation for cobalt. The D4WM detection sensitivity, in terms of the concentration-absorptivity product, is 4.05 X 10(-4) cm-1 for cobalt(II) in ethanol. Our preliminary detection sensitivity compares favorably with other laser-based spectrometric methods. This nonlinear laser technique is applicable to both fluorescing and nonfluorescing analytes.     

Simultaneous detection of single molecules and singulated ensembles of molecules enables immunoassays with broad dynamic range

We report a method for combining the detection of single molecules (digital) and an ensemble of molecules (analog) that is capable of detecting enzyme label from 10(-19) M to 10(-13) M, for use in high sensitivity enzyme-linked immunosorbent assays (ELISA). The approach works by capturing proteins on microscopic beads, labeling the proteins with enzymes using a conventional multistep immunosandwich approach, isolating the beads in an array of 50-femtoliter wells (Single Molecule Array, SiMoA), and detecting bead-associated enzymatic activity using fluorescence imaging. At low concentrations of proteins, when the ratio of enzyme labels to beads is less than ～1.2, beads carry either zero or low numbers of enzymes, and protein concentration is quantified by counting the presence of "on" or "off" beads (digital regime). (1) At higher protein concentrations, each bead typically carries multiple enzyme labels, and the average number of enzyme labels present on each bead is quantified from a measure of the average fluorescence intensity (analog regime). Both the digital and analog concentration ranges are quantified by a common unit, namely, average number of enzyme labels per bead (AEB). By combining digital and analog detection of singulated beads, a linear dynamic range of over 6 orders of magnitude to enzyme label was achieved. Using this approach, an immunoassay for prostate specific antigen (PSA) was developed. The combined digital and analog PSA assay provided linear response over approximately four logs of concentration ([PSA] from 8 fg/mL to 100 pg/mL or 250 aM to 3.3 pM). This approach extends the dynamic range of ELISA from picomolar levels down to subfemtomolar levels in a single measurement.     

Strategies for bioanalysis of an oligonucleotide class macromolecule from rat plasma using liquid chromatography-tandem mass spectrometry

Electrospray ionization (ESI) liquid chromatography-tandem mass spectrometry (LC/MS/MS) assays provide high-throughput and selective methods for quantitation of small molecules. Use of LC/MS/MS assays for macromolecules, like oligonucleotides, is challenging due to lack of sensitivity and low analyte recovery from biomatrixes. Due to this fact, the method of choice for oligonucleotides quantitation remains hybridization-based ligand-binding assays. These biological assays usually possess high sensitivity but low selectivity and narrow dynamic range. They also require optimizing suitable "capture and detection" probes, which can be prohibitively time-consuming and expensive in a drug discovery lead-optimization scenario. In this paper, we present a unique LC/MS/MS assay for a model phosphorothioate backbone oligodeoxynucleotide (ODN) drug (7692 amu) from rat plasma. Multiple analytical challenges were encountered. The strategies used to solve these challenges should prove useful to scientists pursuing mass spectrometry (MS) to quantitate oligonucleotides. The challenges include analyte multiple charging and cation adduction (reduced sensitivity), oxidation of analyte on drying and high protein binding (low recovery), ODN affinity to exposed silica (low chromatographic reproducibility and high carryover), nonspecific binding of analyte to containers (low storage stability), and optimization/synthesis of an appropriate internal standard (interference and cross-talk). A buffer (7 mM triethylamine and 3 mM ammonium formate)/methanol, 50:50 (v/v), was used as an ESI-MS infusion solvent and produced a sharp multiple charge-state distribution. The sample extraction method combined a phenol/chloroform liquid-liquid extraction and solid-phase extraction steps, which improved the absolute recovery to >70%. The method was validated in the range of 5-2000 ng/mL and had precision (percent relative standard deviation)<10.1% and accuracy (percent relative error)<11.4%.     

Integrated affinity capture, purification, and capillary electrophoresis microdevice for quantitative double-stranded DNA analysis

A novel injection method is developed that utilizes a thermally switchable oligonucleotide affinity capture gel to mediate the concentration, purification, and injection of dsDNA for quantitative microchip capillary electrophoresis analysis. The affinity capture matrix consists of a 20 base acrydite modified oligonucleotide copolymerized into a 6% linear polyacrylamide gel that captures ssDNA or dsDNA analyte including PCR amplicons and synthetic oligonucleotides. Double stranded PCR amplicons with complementarity to the capture probe up to 81 bases from their 5' terminus are reproducibly captured via helix invasion. By integrating the oligo capture matrix directly with the CE separation channel, the electrophoretically mobilized target fragments are quantitatively captured and injected after thermal release for unbiased, efficient, and quantitative analysis. The capture process exhibits optimal efficiency at 44 degrees C and 100 V/cm with a 20 microM affinity capture probe (TM = 57.7 degrees C). A dsDNA titration assay with 20 bp fragments validated that dsDNA is captured at the same efficiency as ssDNA. Dilution studies with a duplex 20mer show that targets can be successfully captured and analyzed with a limit of detection of 1 pM from 250 nL of solution (approximately 150,000 fluorescent molecules). Simultaneous capture and injection of amplicons from E. coli K12 and M13mp18 using a mixture of two different capture probes demonstrates the feasibility of multiplex target capture. Unlike the traditional cross-injector, this method enables efficient capture and injection of dsDNA amplicons which will facilitate the quantitative analysis of products from integrated nanoliter-scale PCR reactors.     

Taguchi design-based optimization of sandwich immunoassay microarrays for detecting breast cancer biomarkers

Taguchi design, a statistics-based design of experiment method, is widely used for optimization of products and complex production processes in many different industries. However, its use for antibody microarray optimization has remained underappreciated. Here, we provide a brief explanation of Taguchi design and present its use for the optimization of antibody sandwich immunoassay microarray with five breast cancer biomarkers: CA15-3, CEA, HER2, MMP9, and uPA. Two successive optimization rounds with each 16 experimental trials were performed. We tested three factors (capture antibody, detection antibody, and analyte) at four different levels (concentrations) in the first round and seven factors (including buffer solution, streptavidin-Cy5 dye conjugate concentration, and incubation times for five assay steps) with two levels each in the second round; five two-factor interactions between selected pairs of factors were also tested. The optimal levels for each factor as measured by net assay signal increase were determined graphically, and the significance of each factor was analyzed statistically. The concentration of capture antibody, streptavidin-Cy5, and buffer composition were identified as the most significant factors for all assays; analyte incubation time and detection antibody concentration were significant only for MMP9 and CA15-3, respectively. Interactions between pairs of factors were identified, but were less influential compared with single factor effects. After Taguchi optimization, the assay sensitivity was improved between 7 and 68 times, depending on the analyte, reaching 640 fg/mL for uPA, and the maximal signal intensity increased between 1.8 and 3 times. These results suggest that Taguchi design is an efficient and useful approach for the rapid optimization of antibody microarrays.     

Highly selective detection of single-nucleotide polymorphisms using a quartz crystal microbalance biosensor based on the toehold-mediated strand displacement reaction

Toehold-mediated strand displacement reaction (SDR) is first introduced to develop a simple quartz crystal microbalance (QCM) biosensor without an enzyme or label at normal temperature for highly selective and sensitive detection of single-nucleotide polymorphism (SNP) in the p53 tumor suppressor gene. A hairpin capture probe with an external toehold is designed and immobilized on the gold electrode surface of QCM. A successive SDR is initiated by the target sequence hybridization with the toehold domain and ends with the unfolding of the capture probe. Finally, the open-loop capture probe hybridizes with the streptavidin-coupled reporter probe as an efficient mass amplifier to enhance the QCM signal. The proposed biosensor displays remarkable specificity to target the p53 gene fragment against single-base mutant sequences (e.g., the largest discrimination factor is 63 to C-C mismatch) and high sensitivity with the detection limit of 0.3 nM at 20 °C. As the crucial component of the fabricated biosensor for providing the high discrimination capability, the design rationale of the capture probe is further verified by fluorescence sensing and atomic force microscopy imaging. Additionally, a recovery of 84.1% is obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of employing this biosensor in detecting SNPs in biological samples.