Multifunctionalized Reduced Graphene Oxide Biosensors for Simultaneous Monitoring of Structural Changes in Amyloid-ß 40.

Determination of the conformation (monomer, oligomer, or fibril) of amyloid peptide aggregates in the human brain is essential for the diagnosis and treatment of Alzheimer's disease (AD). Accordingly, systematic investigation of amyloid conformation using analytical tools is essential for precisely quantifying the relative amounts of the three conformations of amyloid peptide. Here, we developed a reduced graphene oxide (rGO) based multiplexing biosensor that could be used to monitor the relative amounts of the three conformations of various amyloid-ß 40 (Aß40) fluids. The electrical rGO biosensor was composed of a multichannel sensor array capable of individual detection of monomers, oligomers, and fibrils in a single amyloid fluid sample. From the performance test of each sensor, we showed that this method had good analytical sensitivity (1 pg/mL) and a fairly wide dynamic range (1 pg/mL to 10 ng/mL) for each conformation of Aß40. To verify whether the rGO biosensor could be used to evaluate the relative amounts of the three conformations, various amyloid solutions (monomeric Aß40, aggregated Aß40, and disaggregated Aß40 solutions) were employed. Notably, different trends in the relative amounts of the three conformations were observed in each amyloid solution, indicating that this information could serve as an important parameter in the clinical setting. Accordingly, our analytical tool could precisely detect the relative amounts of the three conformations of Aß40 and may have potential applications as a diagnostic system for AD.

Study of Alzheimer's Disease-Related Biophysical Kinetics with a Microslit-Embedded Cantilever Sensor in a Liquid Environment.

A microsized slit-embedded cantilever sensor (slit cantilever) was fabricated and evaluated as a biosensing platform in a liquid environment. In order to minimize the degradation caused by viscous damping, a 300 × 100 µm² (length × width) sized cantilever was released by a 5 µm gap-surrounding and vibrated by an internal piezoelectric-driven self-actuator. Owing to the structure, when the single side of the slit cantilever was exposed to liquid a significant quality factor (Q = 35) could be achieved. To assess the sensing performance, the slit cantilever was exploited to study the biophysical kinetics related to Aß peptide. First, the quantification of Aß peptide with a concentration of 10 pg/mL to 1 µg/mL was performed. The resonant responses exhibited a dynamic range from 100 pg/mL to 100 ng/mL (-56.5 to -774 ΔHz) and a dissociation constant (KD) of binding affinity was calculated as 1.75 nM. Finally, the Aß self-aggregation associated with AD pathogenesis was monitored by adding monomeric Aß peptides. As the concentration of added analyte increased from 100 ng/mL to 10 µg/mL, both the frequency shift values (-813 to -1804 ΔHz) and associate time constant increased. These results showed the excellent sensing performance of the slit cantilever overcoming a major drawback in liquid environments to become a promising diagnostic tool candidate.

A Micro-Preconcentrator Combined Olfactory Sensing System with a Micromechanical Cantilever Sensor for Detecting 2,4-Dinitrotoluene Gas Vapor.

Preventing unexpected explosive attacks and tracing explosion-related molecules require the development of highly sensitive gas-vapor detection systems. For that purpose, a micromechanical cantilever-based olfactory sensing system including a sample preconcentrator was developed to detect 2,4-dinitrotoluene (2,4-DNT), which is a well-known by-product of the explosive molecule trinitrotoluene (TNT) and exists in concentrations on the order of parts per billion in the atmosphere at room temperature. A peptide receptor (His-Pro-Asn-Phe-Ser-Lys-Tyr-Ile-Leu-His-Gln-Arg) that has high binding affinity for 2,4-DNT was immobilized on the surface of the cantilever sensors to detect 2,4-DNT vapor for highly selective detection. A micro-preconcentrator (µPC) was developed using Tenax-TA adsorbent to produce higher concentrations of 2,4-DNT molecules. The preconcentration was achieved via adsorption and thermal desorption phenomena occurring between target molecules and the adsorbent. The µPC directly integrated with a cantilever sensor and enhanced the sensitivity of the cantilever sensor as a pretreatment tool for the target vapor. The response was rapidly saturated within 5 min and sustained for more than 10 min when the concentrated vapor was introduced. By calculating preconcentration factor values, we verified that the cantilever sensor provides up to an eightfold improvement in sensing performance.

Multifunctionalized cantilever systems for electronic nose applications.

Multiple target detection using a cantilever is essential for biosensor, chemical sensor, and electronic nose systems. We report a novel microcantilever array chip that includes four microreaction chambers in a chip, which consequently contains four different functionalized surfaces for multitarget detection. For model tests, we designed microcantilever chips and demonstrated the ability of binding of 2,4-dinitrotoluene (DNT) targets onto four different surfaces. We used peptide receptors that are known to have highly selective binding. By simply using four microreaction chambers, we immobilized DNT specific peptide (HPNFSKYILHQRC; SP), DNT nonspecific peptide (TSMLLMSPKHQAC; NSP), and self-assembled monolayer (SAM) as well as a bare cantilever. After flowing DNT gases through the cantilever chip, we could monitor the four different binding signals simultaneously. The shifts in NSP provided information as a negative control because it contained information of temperature fluctuations and mechanical vibration from gas flow. By utilizing the differential signal of the SP and NSP, we acquired 7.5 Hz in resonant responses that corresponds with 160 part per billion (ppb) DNT concentration, showing the exact binding response by eliminating the inevitable thermal noise, vibration noise, as well as humidity effects on the peptide surface.

Highly selective reduced graphene oxide (rGO) sensor based on a peptide aptamer receptor for detecting explosives.

An essential requirement for bio/chemical sensors and electronic nose systems is the ability to detect the intended target at room temperature with high selectivity. We report a reduced graphene oxide (rGO)-based gas sensor functionalized with a peptide receptor to detect dinitrotoluene (DNT), which is a byproduct of trinitrotoluene (TNT). We fabricated the multi-arrayed rGO sensor using spin coating and a standard microfabrication technique. Subsequently, the rGO was subjected to photolithography and an etching process, after which we prepared the DNT-specific binding peptide (DNT-bp, sequence: His-Pro-Asn-Phe-Se r-Lys-Tyr-IleLeu-HisGln-Arg-Cys) and DNT non-specific binding peptide (DNT-nbp, sequence: Thr-Ser-Met-Leu-Leu-Met-Ser-Pro-Lys-His-Gln-Ala-Cys). These two peptides were prepared to function as highly specific and highly non-specific (for the control experiment) peptide receptors, respectively. By detecting the differential signals between the DNT-bp and DNT-nbp functionalized rGO sensor, we demonstrated the ability of 2,4-dinitrotoluene (DNT) targets to bind to DNT-specific binding peptide surfaces, showing good sensitivity and selectivity. The advantage of using the differential signal is that it eliminates unwanted electrical noise and/or environmental effects. We achieved sensitivity of 27 ± 2 × 10-6 per part per billion (ppb) for the slope of resistance change versus DNT gas concentration of 80, 160, 240, 320, and 480 ppm, respectively. By sequentially flowing DNT vapor (320 ppb), acetone (100 ppm), toluene (1 ppm), and ethanol (100 ppm) onto the rGO sensors, the change in the signal of rGO in the presence of DNT gas is 6400 × 10-6 per ppb whereas the signals from the other gases show no changes, representing highly selective performance. Using this platform, we were also able to regenerate the surface by simply purging with N2.

Comparative analyses of plasma amyloid-ß levels in heterogeneous and monomerized states by interdigitated microelectrode sensor system.

Detection of amyloid-ß (Aß) aggregates contributes to the diagnosis of Alzheimer disease (AD). Plasma Aß is deemed a less invasive and more accessible hallmark of AD, as Aß can penetrate blood-brain barriers. However, correlations between biofluidic Aß concentrations and AD progression has been tenuous. Here, we introduce a diagnostic technique that compares the heterogeneous and the monomerized states of Aß in plasma. We used a small molecule, EPPS [4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid], to dissociate aggregated Aß into monomers to enhance quantification accuracy. Subsequently, Aß levels of EPPS-treated plasma were compared to those of untreated samples to minimize inter- and intraindividual variations. The interdigitated microelectrode sensor system was used to measure plasma Aß levels on a scale of 0.1 pg/ml. The implementation of this self-standard blood test resulted in substantial distinctions between patients with AD and individuals with normal cognition (NC), with selectivity and sensitivity over 90%.

An Enhanced Platform to Analyse Low-Affinity Amyloid ß Protein by Integration of Electrical Detection and Preconcentrator.

Sensitivity and limit of detection (LOD) enhancement are essential criteria for the development of ultrasensitive molecular sensors. Although various sensor types have been investigated to enhance sensitivity and LOD, analyte detection and its quantification are still challenging, particularly for protein-protein interactions with low association constants. To solve this problem, here, we used ion concentration polarization (ICP)-based preconcentration to increase the local concentration of analytes in a microfluidic platform for LOD improvement. This was the first demonstration of a microfluidic device with an integrated ICP preconcentrator and interdigitated microelectrode (IME) sensor to detect small changes in surface binding between antigens and antibodies. We detected the amyloid beta (Aß) protein, an Alzheimer's disease marker, with low binding affinity to its antibodies by adopting ICP preconcentration phenomena. We demonstrated that a combination of ICP preconcentrator and IME sensor increased the LOD by 13.8-fold to femtomolar level (8.15 fM), which corresponds to a significant advance for clinical applications.

Enhancing surface functionality of reduced graphene oxide biosensors by oxygen plasma treatment for Alzheimer's disease diagnosis.

We performed oxygen plasma treatment on reduced graphene oxide (rGO) to improve its surface reactivity with respect to biomolecular interactions. Oxygen-plasma-treated rGO surfaces were employed as reactive interfaces for the detection of amyloid-beta (Aß) peptides, the pathological hallmarks of Alzheimer's disease (AD), as the target analytes. By measuring the changes in electrical characteristics and confirmation through topographic analysis, the oxygen-plasma-treated rGO sensors had enhanced surface functionality for better antibody immobilization and sensing performance, with a 3.33-fold steeper slope for the electrical responses versus analyte concentration curve (logarithmic scale) compared to the untreated. The elicited biomolecular reactivity of the rGO surfaces with the oxygen plasma treatment remained at 46-51% of the initial value even after aging for 6h in ambient conditions. This phenomenon was also confirmed by pretreating the rGO surfaces with a blocking agent and subsequently subjecting them to antibody immobilization. Finally, the feasibility of the oxygen-plasma-treated rGO sensors as a diagnostic tool was evaluated with clinical samples of neural-derived exosomal Aß peptides extracted from apparent AD patients and normal controls (NC). In contrast to the untreated sensors (p=0.0460), the oxygen-plasma-treated rGO sensors showed a significant p-value in the identification of clinical samples of AD and NC subjects (p<0.001). These results suggest that oxygen plasma treatment improves sensor performance without complicated fabrication procedures and should aid in the development of novel diagnostic tools based on carbon nanomaterials.

Wafer-scale high-resolution patterning of reduced graphene oxide films for detection of low concentration biomarkers in plasma.

Given that reduced graphene oxide (rGO)-based biosensors allow disposable and repeatable biomarker detection at the point of care, we developed a wafer-scale rGO patterning method with mass productivity, uniformity, and high resolution by conventional micro-electro-mechanical systems (MEMS) techniques. Various rGO patterns were demonstrated with dimensions ranging from 5 µm up to several hundred µm. Manufacture of these patterns was accomplished through the optimization of dry etching conditions. The axis-homogeneity and uniformity were also measured to verify the uniform patternability in 4-inch wafer with dry etching. Over 66.2% of uniform rGO patterns, which have deviation of resistance within range of ±10%, formed the entire wafer. We selected amyloid beta (Aß) peptides in the plasma of APP/PS1 transgenic mice as a study model and measured the peptide level by resistance changes of highly uniform rGO biosensor arrays. Aß is a pathological hallmark of Alzheimer's disease and its plasma concentration is in the pg mL(-1) range. The sensor detected the Aß peptides with ultra-high sensitivity; the LOD was at levels as low as 100 fg mL(-1). Our results provide biological evidences that this wafer-scale high-resolution patterning method can be used in rGO-based electrical diagnostic devices for detection of low-level protein biomarkers in biofluids.