Addressing the use of PDIF-CN2 molecules in the development of n-type organic field-effect transistors for biosensing applications.

BACKGROUND: There is no doubt that future discoveries in the field of biochemistry will depend on the implementation of novel biosensing techniques, able to record biophysiological events with minimal biological interference. In this respect, organic electronics may represent an important new tool for the analysis of structures ranging from single molecules up to cellular events. Specifically, organic field-effect transistors (OFET) are potentially powerful devices for the real-time detection/transduction of bio-signals. Despite this interest, up to date, the experimental data useful to support the development of OFET-based biosensors are still few and, in particular, n-type (electron-transporting) devices, being fundamental to develop highly-performing circuits, have been scarcely investigated. METHODS: Here, films of N,N'-1H,1H-perfluorobutyldicyanoperylene-carboxydi-imide (PDIF-CN2) molecules, a recently-introduced and very promising n-type semiconductor, have been evaporated on glass and silicon dioxide substrates to test the biocompatibility of this compound and its capability to stay electrically-active even in liquid environments. RESULTS: We found that PDIF-CN2 transistors can work steadily in water for several hours. Biocompatibility tests, based on in-vitro cell cultivation, remark the need to functionalize the PDIF-CN2 hydrophobic surface by extra-coating layers (i.e. poly-l-lysine) to favor the growth of confluent cellular populations. CONCLUSIONS: Our experimental data demonstrate that PDIF-CN2 compound is an interesting organic semiconductor to develop electronic devices to be used in the biological field. GENERAL SIGNIFICANCE: This work contributes to define a possible strategy for the fabrication of low-cost and flexible biosensors, based on complex organic complementary metal-oxide-semiconductor (CMOS) circuitry including both p- (hole-transporting) and n-type transistors. This article is part of a Special Issue entitled Organic Bioelectronics-Novel Applications in Biomedicine.

Quantitative biomolecular sensing station based on magnetoresistive patterned arrays.

The combination of magnetoresistive sensors and magnetic labeling of bioanalytes, which are selectively captured by their complementary antibody in the proximity of the sensor is a powerful method in order to attain truly quantitative immunological assays. In this paper we present a technical solution to exploit the existing spin valve technology to readout magnetic signals of bio-functionalized magnetic nanoparticles. The method is simple and reliable, and it is based on a discrete scan of lateral flow strips with a precise control of the contact force between sensor and sample. It is shown that the signal of the sensor is proportional to the local magnetization produced by the nanoparticles in a wide range of concentrations, and the sensitivity thresholds in both calibration samples and real immunorecognition assays of human chorionic gonadotropin hormone are well below the visual inspection limit (5.5 ng/ml). Furthermore the sample scanning approach and the reduced dimensions of the sensors provide unprecedented spatial resolution of the nanoparticle distribution across the supporting nitrocellulose strip, therefore enabling on-stick control references and multi-analyte capability.

Functionalization of carbon buckypaper for the sensitive determination of hydrogen peroxide in human urine.

Here we report on a new approach for the electrochemical detection of hydrogen peroxide (H(2)O(2)) based on the co-immobilization of horseradish peroxidase and methylene blue on the functionalized carbon buckypaper supported by a titanium substrate. Cyclic voltammetry was used to study and optimize the performance of the resulting electrochemical biosensor. The proposed biosensor exhibited high analytical performance towards the quantification of H(2)O(2) at the physiological pH 7.4. Under optimized conditions, the biosensor shows a wide linear response range from 0.1 × 10(-6) to 5 × 10(-4)M concentrations of H(2)O(2). The detection limit was determined to be 7.5 × 10(-8)M (based on S/N=3). Reproducibility and stability of the fabricated biosensor were examined with satisfactory results. The biological relevance of the developed electrochemical biosensor has been further studied by the determination of H(2)O(2) in human urine samples of normal volunteers prior to and following the ingestion of coffee. Increased levels of urinary H(2)O(2) concentration suggest that oxidative stress is induced by coffee drinking in humans. There is considerable interest in oxidative stress as relates to human physiology. The sensitive determination of H(2)O(2) in human urine may serve as a valuable biomarker to effectively elucidate specific levels of oxidative stress in vivo.

Methyl parathion hydrolase based nanocomposite biosensors for highly sensitive and selective determination of methyl parathion.

This article reports the fabrication of a nanocomposite biosensor for the sensitive and specific detection of methyl parathion. The nanocomposite sensing film was prepared via the formation of gold nanoparticles on silica particles, mixing with multiwall carbon nanotubes and subsequent covalent immobilization of methyl parathion hydrolase. The composite of the individual materials was finely tuned to offer the sensing film with high specific surface area and high conductivity. A significant synergistic effect of nanocomposites on the biosensor performance was observed in biosensing methyl parathion. The square wave voltammetric responses displayed well defined peaks, linearly proportional to the concentrations of methyl parathion in the range from 0.001 µg mL⁻¹ to 5.0 µg mL⁻¹ with a detection limit of 0.3 ng mL⁻¹. The application of this biosensor in the analysis of spiked garlic samples was also evaluated. The proposed protocol can be used as a platform for the simple and fast construction of biosensors with good performance for the determination of enzyme-specific electroactive species.

A reagentless, disposable and multiplexed electronic biosensing platform: application to molecular logic gates.

The construction of a reagentless, sensitive, disposable and multiplexed electronic sensing platform for one-spot simultaneous determination of biomolecules with significant difference in size (proteins and small molecules) is described. The sensing surface is fabricated by the hybridization of two types of redox-tags conjugated aptamers with the corresponding complementary DNAs, which are self-assembled on a gold nanoparticle-modified screen printed carbon electrode. The presence of the target analytes leads to the release of the tagged signaling aptamers from the sensing surface, and the surface-remained tags exhibit well-resolved peaks, whose positions and sizes reflect the identities and concentrations of the target analytes, respectively. The application of the proposed sensing platform for molecular logic gate operations is also demonstrated.

Ultra-sensitive biosensor based on mesocellular silica foam for organophosphorous pesticide detection.

A sensitive amperometric acetylcholinesterase (AChE) biosensor was fabricated based on mesocellular silica foam (MSF), which functioned as both an enzyme immobilization matrix and a solid phase extraction (SPE) material for the preconcentration of target molecules. The hydrophilic interface, the good mechanical/chemical stability, and the suitable pore dimension of MSF provided the entrapped AChE a good environment to well maintain its bioactivity at basic condition. The AChE immobilized in MSF showed improved catalytic ability for the hydrolysis of acetylthiocholine, as evidenced by the increasing of the oxidation current of thiocholine, the enzymatic catalytic hydrolysis production of acetylthiocholine. In addition, the MSF with large surface area showed a modest adsorption capacity for monocrotophos, a model organophosphate used in this study, via the hydrogen bond or physical adsorption interaction. The combination of the SPE and the good enzyme immobilization ability in MSF significantly promoted the sensitivity of the biosensor, and the limit of detection has lowered to 0.05 ng/mL. The biosensor exhibited accuracy, good reproducibility, and acceptable stability when used for garlic samples analysis. The strategy may provide a new method to fabricate highly sensitive biosensors for the detection of ultra-trace organophosphorous pesticide infield.

Facile synthesis of Fe(3)O(4)@Al(2)O(3) core-shell nanoparticles and their application to the highly specific capture of heme proteins for direct electrochemistry.

In this study, magnetic core-shell Fe(3)O(4)@Al(2)O(3) nanoparticles (NPs) attached to the surface of a magnetic glassy carbon electrode (MGCE) were used as a functional interface to immobilize several heme proteins including hemoglobin (Hb), myoglobin (Mb) and horseradish peroxidase (HRP) for fabricating protein/Fe(3)O(4)@Al(2)O(3) film. Transmission electron microscope, UV-vis spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry were used to characterize the films. With the advantages of the magnetism and the excellent biocompatibility of the Fe(3)O(4)@Al(2)O(3) NPs, the protein/Fe(3)O(4)@Al(2)O(3) film could be easily fabricated in the present of external magnetic field, and well retained the bioactivity of the immobilized proteins, hence dramatically facilitated direct electron transfer of heme proteins and excellent electrocatalytic behaviors towards H(2)O(2) were demonstrated. The presented system avoids the complex synthesis for protecting Fe(3)O(4) NPs, supplies a facile, low cost and universal way to immobilize proteins, and is promising for construction of third-generation biosensors and other bio-magnetic induction devices.

Design of a sandwich-mode amperometric biosensor for detection of PML/RARα fusion gene using locked nucleic acids on gold electrode.

In this study, a novel DNA electrochemical probe (locked nucleic acid, LNA) was designed and involved in constructing an electrochemical DNA biosensor for detection of promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) fusion gene in acute promyelocytic leukemia for the first time. This biosensor was based on a 'sandwich' sensing mode, which involved a pair of LNA probes (capture probe immobilized at electrode surface and biotinyl reporter probe as an affinity tag for streptavidin-horseradish peroxidase (streptavidin-HRP). Since biotin can be connected with streptavidin-HRP, this biosensor offered an enzymatically amplified electrochemical current signal for the detection of target DNA. In the simple hybridization system, DNA fragment with its complementary DNA fragment was evidenced by amperometric detection, with a detection limit of 74 fM and a linear response range of 0.1-10 pM for synthetic PML/RARα fusion gene in acute promyelocytic leukemia (APL). Otherwise, the biosensor showed an excellent specificity to distinguish the complementary sequence and different mismatch sequences. The new pattern also exhibited high sensitivity and selectivity in mixed hybridization system.

5×5 CMOS capacitive sensor array for detection of the neurotransmitter dopamine.

This work presents miniaturized CMOS (complementary metal oxide semiconductor) capacitive sensors for detection of the neurotransmitter dopamine (DA) down to the sub-fM range. Sensing resolution is significantly enhanced by monolithic sensor integration to reduce the parasitic effect and the use of sub-µm interdigitated microelectrodes as the sensing interface. The 5 × 5 sensor array contains five designs of different electrode sizes and each design has five sensors. The positive charges produced from protonation of boronate and amino group after immobilization of 4-carboxyphenylboronic acid (CPBA) result in an increase of the electrode-analyte capacitance. Then the negative charges produced after binding of CPBA and DA molecules decrease the electrode-analyte capacitance. Signal transduction is achieved through a CMOS readout circuit whose output frequency is inversely proportional to the capacitance. Experimental results showed the ratios of average percentage capacitance changes of the experiment groups over those of the control groups were all larger than one for the five designs at DA concentration of 0.1 fM. Selectivity against the non-analyte species, such as tyramine, has also been demonstrated.

Direct monitoring of pollutants based on an electrochemical biosensor with novel peroxidase (POX1B).

A biosensor for the monitoring of phenolic compounds based on a new protein named POX(1B) purified from garlic which demonstrates similar biochemical properties to peroxidase is investigated. The enzyme was immobilized into chitosan microspheres with covalent link. The properties of the biosensor were analyzed with Fourier transform-infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and cyclic voltammetry (CV). FT-IR demonstrates the covalent attachment of POX(1B) into chitosan and SEM shows high dispersion of the POX(1B) into the chitosan microspheres. The redox potential of POX(1B) in chitosan is 147 mV vs. SCE, which is much higher than reported works using HRP, demonstrating excellent direct electrochemical behaviour of the POX(1B). The electrocatalytic activity of the obtained biosensor towards chlorophenols derivatives in a large range from 10 pM to 10 microM was demonstrated. The mediator free POX(1B)-based biosensor exhibited high sensitivity towards 2,6-dichlorophenol, 4-chlorophenol and pentachlorophenol. A detection limit of 1 pM in the case of 4-chlorophenol was demonstrated with kinetic constant K(m,app) of 0.42 microM with high rapidity of electrochemical response of the biosensor of 1 s.

Multianalyte, dipstick-type, nanoparticle-based DNA biosensor for visual genotyping of single-nucleotide polymorphisms.

DNA biosensors involve molecular recognition of the target sequence by hybridization with specific probes and detection by electrochemical, optical or gravimetric transduction. Disposable, dipstick-type biosensors have been developed recently, which enable visual detection of DNA without using instruments. In this context, we report a multianalyte DNA biosensor for visual genotyping of two single-nucleotide polymorphisms (SNPs). As a model, the biosensor was applied to the simultaneous genotyping of two SNPs, entailing the detection of four alleles. A PCR product that flanks both polymorphic sites is subjected to a single primer extension (PEXT) reaction employing four allele-specific primers, each containing a region complementary to an allele and a characteristic segment that enables subsequent capture on a test zone of the biosensor. The primers are extended with dNTPs and biotin-dUTP only if there is perfect complementarity with the interrogated sequence. The PEXT mixture is applied to the biosensor. As the developing buffer migrates along the strip, all the allele-specific primers are captured by immobilized oligonucleotides at the four test zones of the biosensor and detected by antibiotin-functionalized gold nanoparticles. As a result, the test zones are colored red if extension has occurred denoting the presence of the corresponding allele in the original sample. The excess nanoparticles are captured by immobilized biotinylated albumin at the control zone of the sensor forming another red zone that indicates the proper performance of the system. The assay was applied successfully to the genotyping of twenty clinical samples for two common SNPs of MBL2 gene.

Poly-silicon nanowire field-effect transistor for ultrasensitive and label-free detection of pathogenic avian influenza DNA.

Enhanced surveillance of influenza requires rapid, robust, and inexpensive analytical techniques capable of providing a detailed analysis of influenza virus strains. Functionalized poly-crystalline silicon nanowire field-effect transistor (poly-SiNW FET) was demonstrated to achieve specific and ultrasensitive (at fM level) detection of high pathogenic strain virus (H5 and H7) DNA of avian influenza (AI) which is an important infectious disease and has an immediate need for surveillance. The poly-SiNW FET was prepared by a simple and low-cost method that is compatible with current commercial semiconductor process without expensive E-beam lithography tools for large-scale production. Specific electric changes were observed for AI virus DNA sensing when nanowire surface of poly-SiNW FET was modified with complementary captured DNA probe and target DNA (H5) at fM to pM range could be distinguished. With its excellent electric properties and potential for mass commercial production, poly-SiNW FET can be developed to become a portable biosensor for field use and point-of-care diagnoses.

Antibody-based sensors for heavy metal ions.

Competitive immunoassays for Cd(II), Co(II), Pb(II) and U(VI) were developed using identical reagents in two different assay formats, a competitive microwell format and an immunosensor format with the KinExA 3000. Four different monoclonal antibodies specific for complexes of EDTA-Cd(II), DTPA-Co(II), 2,9-dicarboxyl-1,10-phenanthroline-U(VI), or cyclohexyl-DTPA-Pb(II) were incubated with the appropriate soluble metal-chelate complex. In the microwell assay format, the immobilized version of the metal-chelate complex was present simultaneously in the assay mixture. In the KinExA format, the antibody was allowed to pre-equilibrate with the soluble metal-chelate complex, then the incubation mixture was rapidly passed through a microcolumn containing the immobilized metal-chelate complex. In all four assays, the KinExA format yielded an assay with 10-1000-fold greater sensitivity. The enhanced sensitivity of the KinExA format is most likely due to the differences in the affinity of the monoclonal antibodies for the soluble versus the immobilized metal-chelate complex. The KinExA 3000 instrument and the Cd(II)-specific antibody were used to construct a prototype assay that could correctly assess the concentration of cadmium spiked into a groundwater sample. Mean analytical recovery of added Cd(II) was 114.25+/-11.37%. The precision of the assay was satisfactory; coefficients of variation were 0.81-7.77% and 3.62-14.16% for within run and between run precision, respectively.

A microbial biosensor based on Yarrowia lipolytica for the off-line determination of middle-chain alkanes.

A microbial biosensor based on immobilised psychrotrophic yeast Yarrowia lipolytica integrated to FIA for the determination of middle chain alkanes was developed. The system responded very well to middle chain alkanes even at low operational temperatures down to +5 degrees C. The maximum sensitivity was obtained at 15 degrees C. A linear relationship was observed between the sensor response and dodecane concentration up to 100 microM.