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Low cost paper based immunoassays are receiving interest due to their fast performance and small amounts of biomolecules needed for developing an immunoassay complex. In this work aggregation-induced emissive (AIE) nanoparticles, obtained from a diastereoisomeric mixture of 1,2-di-(4-hydroxyphenyl)-1,2-diphenylethene (TPEDH) in a one-step top-down method, are characterized through Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), and Zeta potential. By measuring the Zeta potential before and after labeling the nanoparticles with antibodies we demonstrate that the colloidal system is stable in a wide pH-range. The AIE-active nanoparticles are deposited on chitosan and glutaraldehyde modified paper pads overcoming the common aggregation-caused quenching (ACQ) effect. Analyte concentrations from 1000ng and below are applied in a model immunocomplex using Goat anti-Rabbit IgG and Rabbit IgG. In the range of 7.81ng-250ng, linear trends with a high R(2) are observed, which leads to a strong increase of the blue fluorescence from the TPEDH nanoparticles.

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Biosensors representing the technological counterpart of living senses have found routine application in amperometric enzyme electrodes for decentralized blood glucose measurement, interaction analysis by surface plasmon resonance in drug development, and to some extent DNA chips for expression analysis and enzyme polymorphisms. These technologies have already reached a highly advanced level and need minor improvement at most. The dream of the "100-dollar" personal genome may come true in the next few years provided that the technological hurdles of nanopore technology or of polymerase-based single molecule sequencing can be overcome. Tailor-made recognition elements for biosensors including membrane-bound enzymes and receptors will be prepared by cell-free protein synthesis. As alternatives for biological recognition elements, molecularly imprinted polymers (MIPs) have been created. They have the potential to substitute antibodies in biosensors and biochips for the measurement of low-molecular-weight substances, proteins, viruses, and living cells. They are more stable than proteins and can be produced in large amounts by chemical synthesis. Integration of nanomaterials, especially of graphene, could lead to new miniaturized biosensors with high sensitivity and ultrafast response. In the future individual therapy will include genetic profiling of isoenzymes and polymorphic forms of drug-metabolizing enzymes especially of the cytochrome P450 family. For defining the pharmacokinetics including the clearance of a given genotype enzyme electrodes will be a useful tool. For decentralized online patient control or the integration into everyday "consumables" such as drinking water, foods, hygienic articles, clothing, or for control of air conditioners in buildings and cars and swimming pools, a new generation of "autonomous" biosensors will emerge.

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Point-of-care (POC) diagnostics brings tests nearer to the site of patient care. The turnaround time is short, and minimal manual interference enables quick clinical management decisions. Growth in POC diagnostics is being continuously fueled by the global burden of cardiovascular and infectious diseases. Early diagnosis and rapid initiation of treatment are crucial in the management of such patients. This review provides the rationale for the use of POC tests in acute coronary syndrome, heart failure, human immunodeficiency virus, and tuberculosis. We also consider emerging technologies that are based on advanced nanomaterials and microfluidics, improved assay sensitivity, miniaturization in device design, reduced costs, and high-throughput multiplex detection, all of which may shape the future development of POC diagnostics.

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OBJECTIVES: We investigated the relationships of biomarkers of various pathophysiologic pathways including high-sensitivity C-reactive protein (hs-CRP), lipocalin-2 (LCN2), myeloperoxidase (MPO) and matrix metalloproteinases 9 (MMP9) with mortality in stroke patients. DESIGN AND METHODS: hs-CRP, LCN2 and MPO concentrations in 92 patients were determined using enzyme-linked immunosorbent assays. MMP9 mRNA concentrations were determined using real-time quantitative reverse transcription-polymerase chain reaction. RESULTS: Twelve patients (13.0%) died at 6 months and 34 patients (37.0%) died at 5 years. The independent predictors for 6-month mortality were hs-CRP (adjusted OR=16.0) and LCN2 (adjusted OR=16.9), while for 5-year mortality was hs-CRP (adjusted OR=5.56). For patients with hs-CRP >3.4 mg/L, an increase in LCN2 was associated with 2.5-fold higher 6-month mortality, while an increase in normalized MMP9 mRNA was associated with 5.8-fold higher 6-month and 1.5-fold higher 5-year mortality. CONCLUSION: hs-CRP was the most significant independent predictor of both short- and long-term mortality after stroke, with LCN2 and MMP9 mRNA each adding further to the risk stratification.

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Advanced multifunctional protein particles encapsulated enzymes and antibodies were developed for enzymatic bioassays and immunoassays with colorimetric and fluorescent channels. A colorimetric channel based on color-substrate precipitation was assigned for enzymatic bioassays for the measurement of hydrogen peroxide with the lowest detectable concentration of 10 µM. A fluorescent channel based on fluorescent labeled antibodies was assigned for immunoassays for the measurement of mouse immunoglobulin G (M IgG) with the lowest detectable concentration of 1.25 µgL(-1). The protein microparticles were fabricated with a template-assisted self-assembly technique termed "Protein Activation Spontaneous Self-assemble" (PASS). The multifunctional protein particles prepared with the PASS method have the advantages of high loading of analytical biomolecules, integrated biological functions, porous structure, and more importantly, they are optically transparent and fluorescence inactive. These unique features make our protein particles a new generation of bead-based platforms to perform enzyme bioassays and immunoassays.

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Two hundreds patients suspected of acute myocardial infarction presenting to the hospital with a median symptom onset of 2.3 h (IQR 1.7-4.0 h) were enrolled in this study. The diagnostic performances of CardioDetect®, a one-step immunotest for heart-type fatty acid-binding protein (H-FABP), and its combination with cardiac troponin I (cTnI) at admission and 2 h after admission, were compared with different cardiac markers. The H-FABP immunotest had better sensitivities (76.6% and 94.4%) than the other cardiac markers and better specificities (88.2% and 81.7%) than myoglobin at admission and 2 h after admission. Both sensitivity and negative predictive value increased to over 90.0% at 2 h after admission. The areas under the receiver operator characteristic curve for the combination of H-FABP with cTnI were the greatest at admission [0.834 (95% CI: 0.774-0.894)].

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The use of rod-like and vesicle-like mesoporous SiO(2) particles for fabricating high performance glucose biosensors is reported. The distinctively high surface areas of mesoporous structures of SiO(2) rendered the adsorption of glucose oxidase (GOx) feasible. Both morphologies of SiO(2) enhanced the sensitivities of glucose biosensors, but by a factor of 36 for vesicle-like SiO(2) and 18 for rod-like SiO(2), respectively. The greater enhancement of vesicle-like SiO(2) can be accounted for by its higher specific surface area (509 m(2)g(-1)) and larger total pore volume (1.49 cm(3)g(-1)). Interestingly, the current responses of GOx immobilized in interior channels of the mesoporous SiO(2) were enhanced much more than those of simple mixtures of GOx and the mesoporous SiO(2). This suggests that the enhancement of current responses arise not only from the high surface area of SiO(2) for high enzyme loading, but also from the improved enzyme activity upon its adsorption on mesoporous SiO(2). Also compared were the performances of glucose biosensors with GOx immobilized on mesoporous SiO(2) by physical adsorption and by covalent binding to 3-aminopropyltrimethoxysilane (APTMS) modified SiO(2) using glutaraldehyde as the cross-linker. The covalent binding approach resulted in higher enzyme loading but lower current sensitivity than with the physical adsorption.

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Recently, it was demonstrated that particles could be utilized as carrier systems for drugs into the hair follicles. In the present study, a two-component drug delivery system is presented consisting of degradable particles loaded with fluorescein isothiocyanate and a separate protease formulation for degradation. The particles were applied alone, 30 min previous to the protease application and simultaneously with the protease onto porcine skin. Subsequently, biopsies were removed, and the penetration depths of the particles were analyzed using laser scanning microscopy. The obtained results demonstrate that the particles alone achieved a penetration depth of around 900 µm. Similar results were obtained for the successive application of particles and protease, whereas a release of the fluorescent dye was only observed in the upper 250 µm corresponding to the penetration depth of the protease. In the case of the simultaneous application, the particles were partly dissolved before application, leading to a reduced particle size and diminished penetration depth. The results revealed that degradable particles are a promising tool for drug delivery into the skin.

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BACKGROUND: Early and accurate diagnosis of rheumatoid arthritis (RA) has become increasingly important. The clinical significance of anti-cyclic citrullinated peptide (CCP) antibody in Chinese RA adults was studied using an anti-CCP2 rapid test. METHODS: Anti-CCP antibody and rheumatoid factor (RF) were determined in 95 RA patients and 140 patients with rheumatic diseases other than RA. RESULTS: Two hundred and thirty five subjects were enrolled in this study. Both sensitivity and specificity of anti-CCP2 ELISA (78.9% & 95.7%) were higher than those of RF (67.4% & 84.3%). The area under the receiver operating characteristic curve for anti-CCP2 ELISA was 0.852 (95% CI: 0.792-0.913) which was larger than that for RF 0.775 (95% CI: 0.710-0.840). Both sensitivity and specificity (75.8% and 92.9%) of the anti-CCP2 rapid test were comparable to the ELISA. However, the sensitivity (62.1%) of a combined strategy by measuring anti-CCP antibody and RF was even lower than either marker alone although the specificity (98.6%) was slightly improved. CONCLUSIONS: Anti-CCP antibody is a valuable tool for diagnosis of RA in Chinese patients. With the use of the reliable and user-friendly anti-CCP rapid test, it may have an important role in the design of therapeutic strategies in RA patients.

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BACKGROUND: Heart-type fatty acid-binding protein (H-FABP) is a heart-specific and highly sensitive biomarker for early diagnosis of acute myocardial infarction (AMI). We investigated the effectiveness of H-FABP for diagnosis of AMI in patients with different ethnic background and different time from symptom onset. METHODS: Venous blood was withdrawn from consecutive patients with acute chest pain admitted to the First Affiliated Hospital of Xinjiang Medical University. The blood samples were used for measurement of creatine kinase MB (CK-MB) and cardiac troponin I (cTnI) using Beckman Coulter DC-800 analyzer, and detection of H-FABP using a one-step bedside immunotest. RESULTS: Two hundred and eighty-nine patients admitted within 12h after the onset of symptoms were recruited in the study. The H-FABP immunotest was found to have higher diagnostic accuracy than cTnI and CK-MB in patients admitted within 3h. The combination of H-FABP and cTnI was found to have the highest diagnostic accuracy (91%) among different cardiac markers and the other combinations. It gave the highest sensitivity [96% (95% CI: 91-98%)] and a comparable specificity [84% (95% CI: 76-89%)] to cTnI alone. CONCLUSION: A cardiac panel consisting of H-FABP and troponin is recommended.

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Using heart-type fatty acid-binding protein (H-FABP) as an early cardiac marker for diagnosis of acute myocardial infarction (AMI) soon after the onset of symptoms requires a rapid assay. A one-step test called, CardioDetect, is used for detection of H-FABP in whole blood sample. Thirty patients suspected of AMI presenting to the emergency department within 12 hours after onset were enrolled in this study. The diagnostic performance of CardioDetect was compared with different cardiac markers. There were 59.1% of patients with positive H-FABP within 6 hours after onset, while there were only 18.2% with positive cardiac troponin I (cTnI). Results indicated the diagnostic power of H-FABP for AMI was significantly higher than that of cTnI. The sensitivity of H-FABP was 81.8%, which was higher than those of the other cardiac markers, while the specificity was comparable. The area under the receiver operating characteristic curve for H-FABP was 0.909, which was significantly larger than the others. With this rapid and sensitive immunotest, H-FABP could be soon introduced in clinical practice in combination with well-established markers like troponins.

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A unique approach of developing a bar code version of lateral-flow enzymatic-based assay for the semi-quantification of hydrogen peroxide is described. The proposed assay system is mainly composed of a goat anti-mouse IgG-horseradish peroxidase conjugate (Gt anti-M IgG-HRP)-coated nitrocellulose (NC) membrane and a peroxidase substrate pad. Unlike the bar code immunochromatographic assay which depends on the stepwise capture of analyte, the principle of enzyme-based bar code lateral-flow assay is based on the different reaction time on successive lines due to the delay in 3,3',5,5'-tetramethylbenzidine (TMB) release. Hydrogen peroxide (H(2)O(2)) acts as a limiting factor which controls the rate of the enzymatic conversion of TMB to blue color complex. The system expresses the concentration of H(2)O(2) in micromole range as three distinct ladder bars in 9 min therefore without the need of any reading device. The major advantages of this assay are its easily readable result, and also its simplicity and low-cost in production offers a cheaper alternative for testing those expensive biosensors might not be available to the third world countries. By incorporating with H(2)O(2)-generating oxidoreductases, the assay can be further extended to detect a variety of analytes with clinical and environmental importance. Glucose was chosen to be the model analyte where the proposed system gave signal response at between 5 microM and 100 microM.

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A lateral-flow, enzyme-based, bar-code assay for creatinine employing the concept of combination of diffusion and kinetics controlled has been developed. Unlike the traditional bar-code version of immunochromatographic assay, which depends on the stepwise capture of colorimetric tracer-labeled antibody-antigen complex by the immobilized antibody on each successive line, the principle of our proposed assay is based on the delay in TMB release and its diffusion in combination with horseradish peroxidase kinetics. Hydrogen peroxide (H(2)O(2)) produced from enzymatic reactions acts as a limiting factor, which controls the rate of conversion of TMB to blue color complex. The assay takes advantage of giving ladder bar result therefore without the need of any reading device. Depending on the amount of enzymes used, the assay can be one (9 min) or two steps (19 min). The strip assay semiquantitatively measures creatinine concentrations ranging from 0 to 400 microM. Thirty urine samples and thirty serum samples were tested, and the assay showed 90.0% and 86.7% agreement compared with conventional Jaffé method, respectively. This assay provides a tool for quick identification of creatinine for patients without the requirement of any instrument.

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Human heart-type fatty acid-binding protein (H-FABP) has a high potential as an early marker for acute myocardial infarction (AMI) being more sensitive than current routine cardiac markers. Seventy-four patients presenting to hospital with a median symptom onset of 2.2 h (IQR 1.5-2.9 h) were enrolled in this study and 54 (73%) had AMI. At presentation, H-FABP gave the highest sensitivity of 83.3% (95% CI: 70.7-92.1) and troponin I (cTnI) gave the highest specificity of 50.0% (95% CI: 27.2-72.8). This study demonstrated that H-FABP immunotest gave a better diagnostic classification at the early stage. Also, AMI was identified significantly earlier by H-FABP than cTnI (17 vs. 6 patients, p<0.05).

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OBJECTIVES: In this study we aimed to investigate the roles of neopterin, C-reactive protein (CRP) and the CRP to neopterin (C/N) ratio to differentiate bacterial from viral aetiology in patients with suspected acute respiratory tract infections (ARTIs) presenting to the emergency department (ED). METHODS: Serum was taken from five hundred and sixty-one patients and used to measure neopterin and CRP levels. The primary outcome was bacterial or viral infection based on positive bacterial culture and positive viral serology. Patients were classified as either: group 1 with positive bacterial culture and mixed bacterial/viral growth; group 2 with virological aetiology, and group 3 with unknown microbiological aetiology. RESULTS: The median of the C/N ratio was 10 times higher in patients with bacterial aetiology than with viral aetiology (12.5 vs 1.2mg/nmol; P<0.0001), and 42 times higher than those in healthy subjects (12.5 vs 0.3mg/nmol; P<0.0001). The area under the receiver-operator characteristic curve for the C/N ratio was 0.840 (0.783-0.898; P<0.05). A cut-off value of "C/N ratio >3" for ruling in/out bacterial/viral infection yielded optimal sensitivity and specificity of 79.5% and 81.5% respectively. A sensitivity analysis performed on all patients (including unknown aetiology) with a cut-off value of "C/N ratio >3" yields a best-case scenario for ruling in/out bacterial/viral infection with sensitivity of 93.1% and specificity of 93.0%. CONCLUSION: This study shows that CRP and neopterin have a role in differentiating bacterial from viral causes of ARTI, and the C/N ratio yields optimal differentiation in the ED setting.

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Biosensing processes such as molecular beacons require non-trivial effort to covalently label or mark biomolecules. We report here a label-free DNA assay system with a simple dye with aggregation-induced emission (AIE) characteristics as the fluorescent bioprobe. 1,1,2,2-Tetrakis[4-(2-bromoethoxy)phenyl]ethene is nonemissive in solution but becomes highly emissive when aggregated. This AIE effect is caused by restriction of intramolecular rotation, as verified by a large increase in the emission intensity by increasing viscosity and decreasing temperature of the aqueous buffer solution of 1,1,2,2-tetrakis[4-(2-triethylammonioethoxy)phenyl]ethene tetrabromide (TTAPE). When TTAPE is bound to a guanine-rich DNA strand (G1) via electrostatic attraction, its intramolecular rotation is restricted and its emission is turned on. When a competitive cation is added to the G1 solution, TTAPE is detached and its emission is turned off. TTAPE works as a sensitive poststaining agent for poly(acrylamide) gel electrophoresis (PAGE) visualization of G1. The dye is highly affinitive to a secondary structure of G1 called the G-quadruplex. The bathochromic shift involved in the G1 folding process allows spectral discrimination of the G-quadruplex from other DNA structures. The strong affinity of TTAPE dye to the G-quadruplex structure is associated with a geometric fit aided by the electrostatic attraction. The distinct AIE feature of TTAPE enables real-time monitoring of folding process of G1 in the absence of any pre-attached fluorogenic labels on the DNA strand. TTAPE can be used as a K+ ion biosensor because of its specificity to K+-induced and -stabilized quadruplex structure.

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In the present study, we describe an InfectCheck barcode-style lateral flow assay for semi-quantitative detection of CRP in distinguishing between bacterial and viral infections. The severity of bacterial infection can be assessed by simply counting the number of red lines developed at the CRP test zone of the test device. If only one visible line appeared at the CRP test zone, it represents a low or mild inflammation with CRP levels <10 mg/L. Two and three visible lines mean moderate (>or=10-25 mg/L) and severe (>or=25-50 mg/L) inflammations respectively while four visible lines stand for a very severe inflammation (>or=50-100 mg/L). If the visible lines become faint and the intensity of the first line is weaker than that of the control line and may even disappear, this outcome corresponds to the stage of having super severe inflammation (>or=100 mg/L). A total of 500 patients admitted to hospital through the Accident and Emergency Unit at the Prince of Wales Hospital were examined. The InfectCheck CRP barcode-style rapid test gave a high sensitivity of 88.7% and a high negative predictive value of 93.8%. This result indicates that the rapid test is reliable to exclude non-infected patients. The calculated intra- and inter-assay coefficient of variation for the five CRP concentration ranges was both within 20.0%. It is a one-step whole blood rapid test without any sample pre-treatment and the result is available within 20 min. This user friendly diagnostic tool can allow self-testing by interested individuals without any expensive reading device.

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This article takes a special focus on signal amplification technologies in immunoassays and new generations of lateral-flow assays. Novel signal amplification technologies based either on new classes of biofunctional nanocrystals consisting of releasable fluorophores or on aggregation-induced emission (AIE) can improve the sensitivity and the limits of detection in immunoassays. A bio-barcode assay also allows signal amplification by utilizing antibody-coated magnetic beads to concentrate the analytes and antibody-coated gold nanoparticle probes to carry with a large number of oligonucleotides. These innovative technologies boost the development of immunoassays. Growth in rapid immunoassay is fueled by the increasing number of diabetics, the globalization of infectious diseases and the surge in cardiovascular and other chronic diseases as well as other chronic conditions. Rapid, near patient, decentralized, point-of-care (POC) tests are emerging as a tool for more efficient diagnosis and patient evaluation. Technological innovations in lateral-flow assays have enabled a move to bring testing closer to the patient. A novel "digital-style" lateral-flow assay provides semi-quantitative results by simply counting the number of red lines in the test without any expensive reading instrument. An immuno-threshold-based assay can give a signal directly proportional to the concentration of a hapten to prevent confusion on interpretation of the test results. In addition, POC tests become more meaningful to healthcare professionals by combining the benefits of new technologies to provide quantitative results. A molecular compact disc provides a high-resolution imaging capability that can identify and quantify many different antigens simultaneously in highly complex immunoassays. Further advances in immunoassays will bring diagnostic testing even closer to the patient, and can help physicians to monitor diseases that require immediate test results, thereby enhancing the quality of patient care.

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This is a first attempt at a brief sketch of the history of biosensors. It is far from complete and rather unsystematic. Many names are still missing, and we apologize for this. But the authors hope to have laid a humble cornerstone for a future "Complete History of Biosensors". We hope that many of our colleagues will contribute!

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