Scalable drop-casting construction of light-addressable photoelectrochemical biosensor on laser-induced graphene electrode arrays for high-throughput drug screening.

A drop-casting method for the scalable construction of a solar cell-type light-addressable photoelectrochemical (PEC) sensor on commercial phenol resin (PR) plates is reported. The sensor was fabricated by laser writing of addressable laser-induced graphene (LIG) electrode arrays on PR plates with ring-disc dual-electrode cell configurations using a 405 nm laser machine. Beneficial from the good hydrophilicity of PR-based LIG and the excellent film formation of bismuth sulfide nanorods (Bi2S3 NRs), uniform Bi2S3 photovoltaic films can be reproducibly deposited onto the LIG disc photoanode array via drop casting modification, which show a sensitive photocurrent response toward thiocholine (TCl) when the ring cathode array was coated with Ag/AgCl. An acetylcholinesterase (AChE)-based PEC biosensor was therefore constructed by a similar drop-casting modification method. The resulting biosensor exhibits good sensitivity toward an AChE inhibitor, i.e., galantamine hydrobromide (GH), with a calibration range of 10-300 µM and a detection limit of 7.33 µM (S/N = 3). Moreover, the biosensor possesses good storage stability, which can achieve the high-throughput screening of AChE inhibitor drugs from traditional Chinese medicines (TCMs). The present work thus demonstrates the promising application of LIG technology in constructing light-addressable PEC sensing devices with high performance and low cost.

Machine Learning-Assisted Automatically Electrochemical Addressable Cytosensing Arrays for Anticancer Drug Screening.

The high-throughput and accurate screening of anticancer drugs is crucial to the preclinical assessment of candidate drugs and remains challenging. Herein, an automatically electrochemical addressable cytosensor (AEAC) for the efficient screening of anticancer drugs is reported. This sensor consists of sectionalized laser-induced graphene arrays decorated by the rhombohedral TiO2 and spherical Pt nanoparticles (LIG-TiO2-Pt) with high electrocatalytic activity for H2O2 and a homemade Ag/Pt electrode couple fixed onto the robot arm. The immobilization of laminin on the surface of LIG-TiO2-Pt can promote its biocompatibility for the growth and proliferation of various tumor cells, which empowers the in situ monitoring of H2O2 directly released from these live cells for drug screening. A machine learning (ML) algorithm is employed to eliminate the possible random or systematic errors of AEAC, realizing rapid, high-throughput, and accurate prediction of different types of anticancer drugs. This ML-assisted AEAC provides a powerful approach to accelerate the evolution of sensing-served tumor therapy.

Disposable solar microcell array-based addressable photoelectrochemical sensor for high-throughput and multiplexed analysis of salivary metabolites.

The high-throughput detection of multiple metabolites in saliva by electrochemical sensors is usually a challenge, which however is essential to the comprehensive evaluation of health status or screening of diseases. Here, a light-addressable and paper-based hydrogen peroxide (H2O2) photoelectrochemical (PEC) sensor for the high-throughput detection of multiple salivary metabolites is reported. This sensor has a unique solar microcell array structure with a silver nanowires/fullerene-Congo red (AgNWs/C60-CR) disc working electrode (WE) and a single-walled carbon nanotubes/platinum nanowires (SWCNTs/PtNWs) ring reference/counter electrode (RE/CE) in each microcell. Enzymes of different metabolites are immobilized on different separated microcells of a cover slide over the sensor, from which enzymatically produced H2O2 can react with p-hydroxyphenyl boric acid (4-HPBA) on the WE of the sensor to generate hydroquinone (HQ) for photocurrent responses. Based on this strategy, a disposable PEC sensor of saliva was developed, which allows the multiplexed detection of uric acid (UA), glucose (GLU) and lactate (LA) in diluted human saliva with high sensitivity and selectivity. Moreover, the detection throughput and application field of the sensor can be easily extended by connecting a series of sensors in parallel or varying the enzymes. The present work thus establishes a cost-effective approach to the scalable construction of versatile biosensing platforms with tunable throughput and varied analytes.

Photosensitive-Stamp-Inspired Scalable Fabrication Strategy of Wearable Sensing Arrays for Noninvasive Real-Time Sweat Analysis.

Wearable sweat sensing is essential to the development of personalized health monitoring in a noninvasive manner with molecular-level insight. Hence, there is an increasing demand for convenient, facile, and efficient fabrication of wearable sensing arrays. Inspired by a photosensitive stamp (PS), we present herein a simple, low-cost, and eco-friendly vacuum filtration-transfer printing method (termed PS-VFTP) for the scalable preparation of single-walled carbon nanotube (SWCNT) based flexible electrode arrays. This method can economically yield customized flexible SWCNT arrays with praiseworthy performance, such as high reproducibility, precision, uniformity, conductivity, and mechanical stability. In addition, the flexible SWCNT arrays can be easily functionalized into high-performance electrochemical sensors for the simultaneous monitoring of sweat metabolites (glucose, lactate) and electrolytes (Na+, K+). The integration of wearable sensing arrays with a signal acquisition and processing circuit system in the intelligent wearable sensors empowers them to realize noninvasive, real-time, and in situ sweat analysis during exercise. More meaningfully, such a PS-VFTP strategy can be easily expanded to the economical manufacturing of other flexible electronic devices.

Light-Addressable Paper-Based Photoelectrochemical Analytical Device with Tunable Detection Throughput for On-Site Biosensing.

Development of biosensing systems resembling optical 96-well plates using portable single-channel electrochemical analyzers is usually a great challenge. Herein, a light-addressable paper-based photoelectrochemical (PEC) analytical device suitable for on-site high-throughput biosensing is reported. This device consists of a solar cell-type single-channel PEC system with plenty of separated detection zones. Each zone contains a silver nanowires/fullerene-Congo red (AgNWs/C60-CR) disc working electrode and a AgNWs ring reference/counter electrode, which can be massively produced by a simple filtration and laser cutting method. Taking advantage of the sensitive photocurrent response of thiocholine (TCl) on AgNWs/C60-CR, an acetylcholinesterase (AChE)-based PEC biosensing system with tunable detection throughput for the on-site screening of ultratrace organophosphorus pesticides (OPs) was established.

Laser-Cut Polymer Tape Templates for Scalable Filtration Fabrication of User-Designed and Carbon-Nanomaterial-Based Electrochemical Sensors.

We report here a simple filtration method for the scalable fabrication of user-designed and carbon-nanomaterial-based electrode arrays using laser-cut poly(vinyl chloride) (PVC) tape templates. This method can produce electrode arrays with high uniformity and low resistance from the dilute dispersions of single-walled carbon nanotubes (SWNTs) and graphene nanoplatelets (GNPs). For these two carbon arrays, the SWNT array is demonstrated to possess several interesting properties, e.g., good mechanical properties, excellent flexibility, and favorable electrochemical behavior. Moreover, its porous structure enables the construction of a paperlike solid-state electrochemical sensor using Nafion electrolytes, which is suitable for the on-site monitoring of trace phenol pollutants in electrolyte-free water. Besides, an electrochemically addressable 36-zone sensor was constructed by this method. With the aid of an inexpensive 3D printer, the addressable sensor can achieve the semiautomatic and high-throughput evaluation of antioxidant capacity on a series of vegetables and fruits using a single-channel electrochemical analyzer.

Portable, Self-Powered, and Light-Addressable Photoelectrochemical Sensing Platforms Using pH Meter Readouts for High-Throughput Screening of Thrombin Inhibitor Drugs.

In this work, a self-powered, portable, and light-addressable photoelectrochemical sensor (P-LAPECS) is developed for efficient drug screening using a handheld pH meter readout. The sensor, which employs thrombin inhibitors as the drug model, is constructed by evenly immobilizing biotin-labeled and thrombin-cleavable peptides on eight separated sensing zones of a single gold film electrode. The incubation of each peptide sensing zone with thrombin leads to the reduction of binding sites for streptavidin-labeled fullerene (C60) PEC bioprobes, which directly reflects the activity of thrombin by the variation of both photocurrent and photovoltage, and therefore allows the screening of thrombin inhibitors using either a single-channel electrochemical analyzer or a portable pH meter. Consequenty, the inhibition efficiency evaluation of multiple thrombin inhibitors can be achieved by just one electrode, and the screening result obtained by the pH meter is very close to that acquired by the electrochemical analyzer. Moreover, P-LAPECS can realize the light-addressable detection of thrombin with a detection limit as low as 0.05 pM. The present work thus demonstrates the possibility of constructing portable, inexpensive, sensitive, and high-throughput biosensing platforms using ubiquitous pH meters for laboratories all over the world.

Label-free and high-throughput biosensing of multiple tumor markers on a single light-addressable photoelectrochemical sensor.

The sensitive and label-free detection of multiple biomarkers on a single electrode by photoelectrochemical (PEC) sensors based on light addressing strategies is very attractive for developing portable and high-throughput biosensing systems. The essential prerequisite of this proposal is the employment of uniform photovoltaic material modified electrodes with high conversion efficiency. Herein, a novel two-step constant potential deposition method for the rapid fabrication of bismuth sulfide film modified ITO electrodes (Bi2S3/ITO) was established. The produced Bi2S3/ITO, with excellent uniformity and high conversion efficiency in visible light ranges, was further modified with gold nanoparticles (AuNPs) and then divided into separated identical sensing zones by insulative paints. The adsorption-based immobilization of antibodies of three tumor markers, i.e., a-fetoprotein (AFP), carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA19-9), onto different sensing zones of the electrode and the further blocking with BSA established a label-free and light-addressable PEC sensor (LF-LAPECS), which can achieve the rapid and sensitive detection of these biomarkers with wide linear ranges, low detection limits and self-calibration ability. Moreover, the detection throughput can be conveniently improved by enlarging the size of the substrate electrode and increasing the number of separated sensing zones. The present work thus demonstrates the promising applications of PEC techniques for developing sensitive, time-saving, cost-effective and high-throughput biosensing methods.

Fully Converting Graphite into Graphene Oxide Hydrogels by Preoxidation with Impure Manganese Dioxide.

Potassium permanganate (KMnO4) has been proved to be an efficient oxidant for converting graphite into graphite oxide, but its slow diffusion in the interlayer of graphite seriously restricts the production of graphene oxide (GO). Here, we demonstrate that the preoxidation of graphite by impure manganese dioxide (MnO2) in a mixture of concentrated sulfuric acid (H2SO4) and phosphorus pentoxide (P2O5) can efficiently improve the synthesis of GO when KMnO4 is employed as the oxidant. The prepared honey-like GO hydrogels possess a high yield of single-layer sheets, large sizes (average lateral size up to 20 µm), wide ranges of stable dispersion concentrations (from dilute solutions, viscous hydrogels, to dry films), and good conductivity after reduction (~2.9 × 10(4) S/m). The mechanism for the improved synthesis of GO by impure MnO2 was explored. The enhanced exfoliation and oxidation of graphite by oxidative Mn ions (mainly Mn(3+)), which are synergistically produced by the reaction of impure MnO2 with H2SO4 and P2O5, are found to be responsible for the improved synthesis of such GO hydrogels. Particularly, preoxidized graphite (POG) can be partially dispersed in water with sonication, which allows the facile construction of flexible and highly conductive graphene nanosheet film electrodes with excellent electrochemical sensing properties.

Ultrasensitive photoelectrochemical biosensing of multiple biomarkers on a single electrode by a light addressing strategy.

Ultrasensitive multiplexed detection of biomarkers on a single electrode is usually a great challenge for electrochemical sensors. Here, a light addressable photoelectrochemical sensor (LAPECS) for the sensitive detection of multiple DNA biomarkers on a single electrode was reported. The sensor was constructed through four steps: (1) immobilization of capture DNA (C-DNA) of different targets on different areas of a single large-sized gold film electrode, (2) recognition of each target DNA (T-DNA) and the corresponding biotin-labeled probe DNA (P-DNA) through hybridization, (3) reaction of the biotin-labeled probe DNA with a streptavidin-labeled all-carbon PEC bioprobe, and (4) PEC detection of multiple DNA targets one by one via a light addressing strategy. Through this principle, the LAPECS can achieve ultrasensitive detection of three DNA sequences related to hepatitis B (HBV), hepatitis C (HCV) and human immunodeficiency (HIV) viruses with a similar wide calibration range of 1.0 pM ∼ 0.01 µM and a low detection limit of 0.7 pM by using one kind of PEC bioprobe. Moreover, the detection throughput of LAPECS may be conveniently expanded by simply enlarging the size of the substrate electrode or reducing the size of the sensing arrays and the light beam. The present work thus demonstrates the promising applications of LAPECS in developing portable, sensitive, high-throughput, and cost-effective biosensing systems.

3D nitrogen-doped graphene/ß-cyclodextrin: host-guest interactions for electrochemical sensing.

Host-guest interactions, especially those between cyclodextrins (CDs, including α-, ß- and γ-CD) and various guest molecules, exhibit a very high supramolecular recognition ability. Thus, they have received considerable attention in different fields. These specific interactions between host and guest molecules are promising for biosensing and clinical detection. However, there is a lack of an ideal electrode substrate for CDs to increase their performance in electrochemical sensing. Herein, we propose a new 3D nitrogen-doped graphene (3D-NG) based electrochemical sensor, taking advantage of the superior sensitivity of host-guest interactions. Our 3D-NG was fabricated by a template-directed chemical vapour deposition (CVD) method, and it showed a large specific surface area, a high capacity for biomolecules and a high electron transfer efficiency. Thus, for the first time, we took 3D-NG as an electrode substrate for ß-CD to establish a new type of biosensor. Using dopamine (DA) and acetaminophen (APAP) as representative guest molecules, our 3D-NG/ß-CD biosensor shows extremely high sensitivities (5468.6 µA mM(-1) cm(-2) and 2419.2 µA mM(-1) cm(-2), respectively), which are significantly higher than those reported in most previous studies. The stable adsorption of ß-CD on 3D-NG indicates potential applications in clinical detection and medical testing.

A three-dimensional nitrogen-doped graphene structure: a highly efficient carrier of enzymes for biosensors.

In recent years, graphene-based enzyme biosensors have received considerable attention due to their excellent performance. Enormous efforts have been made to utilize graphene oxide and its derivatives as carriers of enzymes for biosensing. However, the performance of these sensors is limited by the drawbacks of graphene oxide such as slow electron transfer rate, low catalytic area and poor conductivity. Here, we report a new graphene-based enzyme carrier, i.e. a highly conductive 3D nitrogen-doped graphene structure (3D-NG) grown by chemical vapour deposition, for highly effective enzyme-based biosensors. Owing to the high conductivity, large porosity and tunable nitrogen-doping ratio, this kind of graphene framework shows outstanding electrical properties and a large surface area for enzyme loading and biocatalytic reactions. Using glucose oxidase (GOx) as a model enzyme and chitosan (CS) as an efficient molecular binder of the enzyme, our 3D-NG based biosensors show extremely high sensitivity for the sensing of glucose (226.24 µA mM(-1) m(-2)), which is almost an order of magnitude higher than those reported in most of the previous studies. The stable adsorption and outstanding direct electrochemical behaviour of the enzyme on the nanocomposite indicate the promising application of this 3D enzyme carrier in high-performance electrochemical biosensors or biofuel cells.

Rapid in situ detection of ultratrace 2,4-dinitrotoluene solids by a sandwiched paper-like electrochemical sensor.

This work reported the rapid in situ detection of ultratrace 2,4-dinitrotoluene (DNT) solids on various substrates by a sandwiched paper-like electrochemical sensor. The sensor, prepared by a simple electroless deposition method without using special instruments, possessed a unique thin-film structure of an insulated polyvinylidene fluoride (PVDF) membrane in between two gold (Au) conducting layers. The resulting gold-PVDF sandwich (GPVDFS) array exhibited excellent flexibility, porosity and electrochemical performance as a highly integrated dual-electrode sensor platform. The infiltration of nonvolatile ionic liquid (IL) electrolytes containing ferrocene (Fc) into the GPVDFS array produced a paper-like electrochemical sensor, which can directly detect ultratrace DNT solids on various substrate surfaces (e.g., plant leaves, gloves and metal knives) with detection limit as low as 0.33 ng/mm(2). The critical role of Fc in the detection of DNT at this dual-electrode sensor was explored. The compensating electrochemical oxidation of Fc at the counter/reference electrode was found to be essential to the reduction of DNT at the working electrode when IL electrolytes were employed. The present work thus demonstrated the promising applications of paper-based porous electrode arrays in developing IL-based electrochemical sensors for the in situ detection of analyte solids in complicated environments.

Preparation and application of a novel electrochemical sensing material based on surface chemistry of polyhydroquinone.

A new analogue of polydopamine (PDA), i.e., polyhydroquinone (PH2Q), was polymerized and its surface chemistry was studied by different ways of characterization. PH2Q was produced by the self-polymerization of H2Q mediated by dissolved oxygen, and the self-polymerization process was strongly dependent on the type and the pH value of the buffer solutions. PH2Q can not only achieve surface hydrophilization of different substrates like polyethylene terephthalate (PET) film, graphite strip, C12SH/Au and wax slice, but also possess several unique properties like reversible adsorption, good solubility and low cost. These properties made PH2Q an ideal polymeric modifier for the noncovalent functionalization of some nanomaterials. By simply grinding with PH2Q, pristine multi-walled carbon nanotubes (MWNTs) can be readily dispersed in water with high solubility and good stability. The resulting MWNT-PH2Q composite exhibited excellent electrochemical performance, which was employed for the simultaneous determination of dopamine (DA) and uric acid (UA).

Ultrasensitive all-carbon photoelectrochemical bioprobes for zeptomole immunosensing of tumor markers by an inexpensive visible laser light.

A novel enzyme-free and all-carbon photoelectrochemical (PEC) bioprobe, based on carboxylated multiwalled carbon nanotube-Congo red-fullerene nanohybrids (MWNTCOOH-CR-C60), for the ultrasensitive immunosensing of carcinoembryonic antigen (CEA) was reported. The MWNTCOOH-CR-C60 nanohybrids, prepared by mechanically grinding a mixture of MWNTCOOH, C60, and CR at a certain mass ratio, had good water dispersibility and high PEC conversion efficiency in visible light ranges. Covalent binding of the detection antibody of CEA on the MWNTCOOH-CR-C60 nanohybrids produced a sensitive PEC bioprobe for detection of CEA by sandwich immunosensing. The corresponding immunosensor, employing an inexpensive and portable green laser light, possessed a wide calibration range of 1.0 pg/mL~100.0 ng/mL and a low detection limit of 0.1 pg/mL (calculated 5 zmol for a 10.0 µL sample solution) (S/N = 3), which was successfully applied to the detection of CEA in serum samples from both healthy people and cancer patients. The present work thus demonstrated the promising application of fullerene-based nanocomposites in developing highly sensitive, environmentally friendly, and cost-effective PEC biosensors.

Capillary microextraction combined with fluorinating assisted electrothermal vaporization inductively coupled plasma optical emission spectrometry for the determination of trace lanthanum, europium, dysprosium and yttrium in human hair.

In this work, a congo red modified single wall carbon nanotubes (CR-SWCNTs) coated fused-silica capillary was prepared and used for capillary microextraction (CME) of trace amounts of lanthanum (La), europium (Eu), dysprosium (Dy) and yttrium (Y) in human hair followed by fluorinating assisted electrothermal vaporization-inductively coupled plasma-optical emission spectrometry (FETV-ICP-OES) determination. The adsorption properties and stability of the prepared CR-SWCNTs coated capillary along with the various factors affecting the separation/preconcentration of La, Eu, Dy and Y by CME were investigated in detail. Under the optimized conditions, with a consumption of 2 mL sample solution, a theoretical enrichment factor of 50 and a detection limit (3σ) of 0.12 ng mL(-1) for La, 0.03 ng mL(-1) for Eu, 0.11 ng mL(-1) for Dy and 0.03 ng mL(-1) for Y were obtained, respectively. The preparation reproducibility of the CR-SWCNTs coated capillary was investigated and the relative standard deviations (RSDs) were ranging from 4.1% (Eu) to 4.4% (La) (CLa, Dy=1.4 ng mL(-1); CY, Eu=0.25 ng mL(-1), n=7) in one batch, and from 5.7% (Eu) to 6.1% (Y) (CLa, Dy=1.4 ng mL(-1); CY, Eu=0.25 ng mL(-1), n=5) among different batches. The proposed method was applied to the analysis of real-world human hair sample and the recoveries for the spiked sample were in the range of 93-105%. The method was also applied to the determination of La, Eu, Dy and Y in Certified Reference Material of GBW07601 human hair, and the determined values were in good agreement with the certified values.

Inkjet printing of nanoporous gold electrode arrays on cellulose membranes for high-sensitive paper-like electrochemical oxygen sensors using ionic liquid electrolytes.

A simple approach to the mass production of nanoporous gold electrode arrays on cellulose membranes for electrochemical sensing of oxygen using ionic liquid (IL) electrolytes was established. The approach, combining the inkjet printing of gold nanoparticle (GNP) patterns with the self-catalytic growth of these patterns into conducting layers, can fabricate hundreds of self-designed gold arrays on cellulose membranes within several hours using an inexpensive inkjet printer. The resulting paper-based gold electrode arrays (PGEAs) had several unique properties as thin-film sensor platforms, including good conductivity, excellent flexibility, high integration, and low cost. The porous nature of PGEAs also allowed the addition of electrolytes from the back cellulose membrane side and controllably produced large three-phase electrolyte/electrode/gas interfaces at the front electrode side. A novel paper-based solid-state electrochemical oxygen (O(2)) sensor was therefore developed using an IL electrolyte, 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF(6)). The sensor looked like a piece of paper but possessed high sensitivity for O(2) in a linear range from 0.054 to 0.177 v/v %, along with a low detection limit of 0.0075% and a short response time of less than 10 s, foreseeing its promising applications in developing cost-effective and environment-friendly paper-based electrochemical gas sensors.

A disposable electrochemical sensor for the determination of indole-3-acetic acid based on poly(safranine T)-reduced graphene oxide nanocomposite.

A disposable electrochemical sensor for the determination of indole-3-acetic acid (IAA) based on nanocomposites of reduced graphene oxide (rGO) and poly(safranine T) (PST) was reported. The sensor was prepared by coating a rGO film on a pre-anodized graphite electrode (AGE) through dipping-drying and electrodepositing a uniform PST layer on the rGO film. Scanning electron microscopic (SEM) and infrared spectroscopic (IR) characterizations indicated that PST-rGO formed a rough and crumpled composite film on AGE, which exhibited high sensitive response for the oxidation of IAA with 147-fold enhancement of the current signal compared with bare AGE. The voltammetric current has a good linear relationship with IAA concentration in the range 1.0×10(-7)-7.0×10(-6)M, with a low detection limit of 5.0×10(-8)M. This sensor has been applied to the determination of IAA in the extract samples of several plant leaves and the recoveries varied in the range of 97.71-103.43%.

Fabrication and application of poly(alizarin red S)-carbon nanotubes composite film based nitrite sensor.

In this work, a novel electrochemical nitrite sensor for sensitive determination of nitrite based on poly(alizarin red S)-multi-wall carbon nanotubes (PARS-MWNTs) composite film on the glassy carbon electrode was described. The surface morphologies of different electrodes were characterized by scanning electron microscopy. Cyclic voltammetry, chronocoulometry and linear sweep voltammetry were used to investigate the electrochemical response and oxidation mechanism of nitrite at the PARS-MWNTs composite film based sensor. The experimental parameters were optimized, such as electropolymerization pH value, film thickness and detection pH value et al. Under optimal working conditions, the oxidation peak current of nitrite linearly increased with its concentration in the range of 30 nM to 1.1 mM with a low detection limit of 2 nM. The PARS-MWNTs composite film based nitrite sensor was applied to the determination of nitrite in sausage, and the good recovery indicated that it may have practical applications in nitrite monitoring system.

Electrochemical behavior of biocatalytical composite based on heme-proteins, didodecyldimethylammonium bromide and room-temperature ionic liquid.

A novel biocompatible composite film based on a water-insoluble surfactant, didodecyldimethylammonium bromide (DDAB), and a hydrophobic room-temperature ionic liquid (RTIL), 1-hexyl-3-methyl-imidazolium hexafluorophosphate (HIMIMPF(6)), for the immobilization of biocatalytical proteins was reported. Differential scanning calorimetry (DSC) showed that the DDAB-HIMIMPF(6) composite film has higher thermal stability than the DDAB film alone. SEM images indicated that different microstructures existed between the DDAB film and the composite film, indicating the interaction between DDAB and RTILs. This composite can be used as the immobilization matrix of proteins and other biomacromolecules. Heme-proteins, including hemoglobin (Hb), myoglobin (Mb) and horseradish peroxidase (HRP), were used as model proteins for studying the electrochemical behaviors of the resulting biocatalytical composite films. In the case of Hb, a pair of well-defined quasi-reversible redox peaks was obtained when the composite film containing Hb was modified on a glassy carbon electrode. The formal potential (E degrees '), the surface coverage (Gamma(*)) and the electron transfer rate constant (k(s)) were calculated as -0.308V, 1.32x10(-11)molcm(-2) and 11.642s(-1), respectively. While, these parameters for Hb on DDAB films alone were -0.309V, 7.20x10(-12)molcm(-2) and 2.748s(-1), respectively. Therefore, the composite are more suitable for the direct electron transfer between Hb than DDAB alone. The native conformation and bioactivity of Hb adsorbed on the composite film was proved to be maintained, reflected by the unchanged ultraviolet-visible (UV-vis) as well as the catalytic activity toward hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) compared with the free Hb molecules. Furthermore, Hb on the composite film are more sensitive for the detection of hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) than that on DDAB film alone. The linear range of H(2)O(2) on Hb/DDAB-RTILs/GC electrode is from 0.5 to 57.5microM with linear regression equations I(microA)=0.149+0.00904C(microM), while, the linear range of H(2)O(2) on Hb/DDAB/GC electrode is from 0.5 to 57.5microM with linear regression equations I(microA)=0.0938+0.00553C(microM). For NO, its linear range on Hb/DDAB-RTILs/GC electrode is from 1.8 to 21.6microM with linear regression equations I(microA)=0.0937+0.0232C(microM). But its linear range on Hb/DDAB/GC electrode is from 1.8 to 21.6microM with linear regression equations I(microA)=0.0285+0.0167C(microM). Similar results were observed for Mb and HRP in the DDAB-HIMIMPF(6) composite film.