Sensitive, rapid and quantitative detection of substance P in serum samples using an integrated microfluidic immunochip.

Miniaturized diagnostic devices hold the promise of accelerate the specific and sensitive detection of various biomarkers, which can translate into many areas of medicine - from cheaper clinical trials, to early diagnosis and treatment of complex diseases. Therefore, we report on a disposable integrated chip-based capillary immunoassay featuring a microfluidic ELISA format combining electrochemical detection and low-cost fabrication employing a dry film photoresist, Vacrel(®) 8100. The readily accessible carboxylate groups on the material surface allow fast and high yield immobilization of biomolecules using amine-specific coupling via reactive esters requiring no laborious surface pretreatment. The integrated microfluidic system provides a convenient platform for a flow-through immunoassay. Capillary force is used for easy reagent delivery and loading the chip channel. We performed rapid quantification of serum level of substance P, a potential biomarker of acute neuroinflammation, using the developed microfluidic immunochip. Our miniaturized assay demonstrated a sensitive electrochemical detection of the antigen at 15.4pgml(-1) (11.5pM) using only 5µl of the biological fluid while cutting the total assay preparation time in half and the read-out time to 10min. Combining microfluidics and fabrication suitable for mass production with the capability of testing clinically relevant samples creates conditions for the construction of low-cost and portable point of care diagnostic devices with minimal auxiliary electronics.

A micro-cantilever sensor chip based on contact angle analysis for a label-free troponin I immunoassay.

Cantilever sensors have been extensively explored as a promising technique for real-time and label-free analyses in biological systems. A major sensing principle utilized by state-of-the-art cantilever sensors is based on analyte-induced surface stress changes, which result in static bending of a cantilever. The sensor performance, however, suffers from the intrinsically small change in surface stress induced by analytes, especially for molecular recognition such as antigen-antibody binding. Through the contact angle change on a tailored solid surface, it is possible to convert a tiny surface stress into a capillary force-a much larger physical quantity needed for a practical sensor application. In this work, a micro-cantilever sensor based on contact angle analysis (CAMCS) was proposed to effectively enhance the sensitivity of a sensor in proportion to the square of the length to thickness ratio of the cantilever structure. CAMCS chips were fabricated using a standard complementary-metal-oxide-semiconductor (CMOS) process to demonstrate a 1250-fold enhancement in the sensitivity of surface stress to bioanalyte adsorption using a piezoresistive sensing method. A real-time and label-free troponin I (cTnI) immunoassay, which is now widely used in clinics and considered a gold standard for the early diagnosis and prognosis of cardiovascular disease, was performed to demonstrate cTnI detection levels as low as 1 pg mL(-1). The short detection time of this assay was within several minutes, which matches the detection time of commercially available instruments that are based on fluorescence-labeling techniques.

Structural framework for covalent inhibition of Clostridium botulinum neurotoxin A by targeting Cys165.

Clostridium botulinum neurotoxin type A (BoNT/A) is one of the most potent toxins for humans and a major biothreat agent. Despite intense chemical efforts over the past 10 years to develop inhibitors of its catalytic domain (catBoNT/A), highly potent and selective inhibitors are still lacking. Recently, small inhibitors were reported to covalently modify catBoNT/A by targeting Cys(165), a residue located in the enzyme active site just above the catalytic zinc ion. However, no direct proof of Cys(165) modification was reported, and the poor accessibility of this residue in the x-ray structure of catBoNT/A raises concerns about this proposal. To clarify this issue, the functional role of Cys(165) was first assessed through a combination of site-directed mutagenesis and structural studies. These data suggested that Cys(165) is more involved in enzyme catalysis rather than in structural property. Then by peptide mass fingerprinting and x-ray crystallography, we demonstrated that a small compound containing a sulfonyl group acts as inhibitor of catBoNT/A through covalent modification of Cys(165). The crystal structure of this covalent complex offers a structural framework for developing more potent covalent inhibitors catBoNT/A. Other zinc metalloproteases can be founded in the protein database with a cysteine at a similar location, some expressed by major human pathogens; thus this work should find broader applications for developing covalent inhibitors.

Substrate-selective supramolecular tandem assays: monitoring enzyme inhibition of arginase and diamine oxidase by fluorescent dye displacement from calixarene and cucurbituril macrocycles.

A combination of moderately selective host-guest binding with the impressive specificity of enzymatic transformations allows the real-time monitoring of enzymatic reactions in a homogeneous solution. The resulting enzyme assays ("supramolecular tandem assays") exploit the dynamic binding of a fluorescent dye with a macrocyclic host in competition with the binding of the substrate and product. Two examples of enzymatic reactions were investigated: the hydrolysis of arginine to ornithine catalyzed by arginase and the oxidation of cadaverine to 5-aminopentanal by diamine oxidase, in which the substrates have a higher affinity to the macrocycle than the products ("substrate-selective assays"). The depletion of the substrate allows the fluorescent dye to enter the macrocycle in the course of the enzymatic reaction, which leads to the desired fluorescence response. For arginase, p-sulfonatocalix[4]arene was used as the macrocycle, which displayed binding constants of 6400 M(-1) with arginine, 550 M(-1) with ornithine, and 60,000 M(-1) with the selected fluorescent dye (1-aminomethyl-2,3-diazabicyclo[2.2.2]oct-2-ene); the dye shows a weaker fluorescence in its complexed state, which leads to a switch-off fluorescence response in the course of the enzymatic reaction. For diamine oxidase, cucurbit[7]uril (CB7) was used as the macrocycle, which showed binding constants of 4.5 x 10(6) M(-1) with cadaverine, 1.1 x 10(5) M(-1) with 1-aminopentane (as a model for the thermally unstable 1-aminopentanal), and 2.9 x 10(5) M(-1) with the selected fluorescent dye (acridine orange, AO); AO shows a stronger fluorescence in its complexed state, which leads to a switch-on fluorescence response upon enzymatic oxidation. It is demonstrated that tandem assays can be successfully used to probe the inhibition of enzymes. Inhibition constants were estimated for the addition of known inhibitors, i.e., S-(2-boronoethyl)-L-cysteine and 2(S)-amino-6-boronohexanoic acid for arginase and potassium cyanide for diamine oxidase. Through the sequential coupling of a "product-selective" with a "substrate-selective" assay it was furthermore possible to monitor a multistep biochemical pathway, namely the decarboxylation of lysine to cadaverine by lysine decarboxylase followed by the oxidation of cadaverine by diamine oxidase. This "domino tandem assay" was performed in the same solution with a single reporter pair (CB7/AO).

Label-free continuous enzyme assays with macrocycle-fluorescent dye complexes.

We introduce a new economic, convenient and general assay principle based on the reversible interaction of water-soluble macrocycles and fluorescent dyes. We show that amino acid decarboxylase activity can be continuously monitored by measuring changes in fluorescence, which result from the competition of the enzymatic product and the dye for forming a complex with a cucurbituril or calixarene macrocycle. The new assay provides a complementary method to the use of antibodies, radioactive markers and labeled substrates.

Analysis of host-assisted guest protonation exemplified for p-sulfonatocalix[4]arene--towards enzyme-mimetic pKa shifts.

The pD dependence of the complexation of p-sulfonatocalix[4]arene (CX4) with the azoalkanes 2,3-diazabicyclo[2.2.1]hept-2-ene (1), 2,3-diazabicyclo[2.2.2]oct-2-ene (2), 2,3-diazabicyclo[2.2.3]non-2-ene (3), and 1-methyl-4-isopropyl-2,3-diazabicyclo[2.2.2]oct-2-ene (4) in D(2)O has been studied. The pD-dependent binding constants, determined by (1)H NMR spectroscopy, were analyzed according to a seven-state model, which included the CX4 tetra- and penta-anions, the protonated and unprotonated forms of the azoalkanes, the corresponding complexes, as well as the complex formed between CX4 and the deuteriated hydronium ion. The variation of the UV absorption spectra, namely the hypsochromic shift in the near-UV band of the azo chromophore upon protonation, was analyzed according to a four-state model. Measurements by independent methods demonstrated that complexation by CX4 shifts the pK(a) values of the guest molecules by around 2 units, thereby establishing a case of host-assisted guest protonation. The pK(a) shift can be translated into improved binding (factor of 100) of the protonated guest relative to its unprotonated form as a result of the cation-receptor properties of CX4. The results are discussed in the context of supramolecular catalytic activity and the pK(a) shifts induced by different types of macrocyclic hosts are compared.

Binding of inorganic cations by p-sulfonatocalix[4]arene monitored through competitive fluorophore displacement in aqueous solution.

A new working principle for detecting inorganic cation binding by water-soluble calix[4]arenes involves the displacement of a fluorescent azoalkane as guest. Fluorescence regeneration is observed for various metal ions, and binding of monovalent cations (alkali and ammonium) to p-sulfonatocalix[4]arene is detected and quantified for the first time.

Spherical shape complementarity as an overriding motif in the molecular recognition of noncharged organic guests by p-sulfonatocalix[4]arene: complexation of bicyclic azoalkanes.

[reaction: see text] The complexation of p-sulfonatocalix[4]arene (CX4) with 2,3-diazabicyclo[2.2.1]hept-2-ene (1), 2,3-diazabicyclo[2.2.2]oct-2-ene (2), 2,3-diazabicyclo[3.2.2]non-2-ene (3), 1-methyl-4-isopropyl-2,3-diazabicyclo[2.2.2]oct-2-ene (4), and 1-phenyl-2,3-diazabicyclo[2.2.2]oct-2-ene (5) was studied in D2O at pD 7.4 by 1H NMR spectroscopy. The formation of deep inclusion complexes was indicated by large upfield 1H NMR shifts of the guest protons (up to 2.6 ppm), which were also used to assign, in combination with 2D ROESY spectra, a preferential inclusion of the isopropyl group of 4 and a dominant inclusion of the azo bicyclic residue for 5. The bicyclic azoalkanes 1-3 showed exceptionally high binding constants on the order of 1000 M(-1), 1-2 orders of magnitude larger than for previously investigated noncharged organic guest molecules. The strong binding was attributed to the spherical shape complementarity between the guest and the conical cavity offered by CX4. Interestingly, although the derivatives 4 and 5 are more hydrophobic, they showed a 2-3 times weaker binding, which was again attributed to the deviation from spherical shape in these bridgehead-substituted derivatives. The preferential inclusion of the hydrophilic but spherical bicyclic residue of 5 rather than the hydrophobic aromatic phenyl group provides a unique observation in aqueous host-guest chemistry and corroborates the pronounced spherical shape affinity of CX4.

Effect of temperature, cholesterol content, and antioxidant structure on the mobility of vitamin E constituents in biomembrane models studied by laterally diffusion-controlled fluorescence quenching.

Kinetic parameters relevant for the antioxidant activity of the vitamin E constituents (alpha, beta, gamma, and delta homologues of tocopherols and tocotrienols) and of an amphiphilic vitamin C derivative, l-ascorbyl 6-palmitate, were determined. Fluorescence quenching experiments of 2,3-diazabicyclo[2.2.2]oct-2-ene in homogeneous acetonitrile-water mixtures afforded reactivity trends in terms of intermolecular quenching rate constants, while the quenching of Fluorazophore-L in liposomes provided the lateral diffusion coefficients relevant for understanding their biological activity in membranes. The reactivity in homogeneous solution was not influenced by the nature of the isoprenoid tail (tocopherol versus tocotrienol), but was dependent on the methylation pattern. The resulting order (alpha > beta = gamma > delta) was found to be in line with their reactivities toward peroxyl radicals as well as the phenolic O-H bond dissociation energies. The mutual lateral diffusion coefficient in POPC liposomes was the same, within error, for different tocopherols and tocotrienols (D(L) = (1.6 +/- 0.2) x 10(-7) cm(2) s(-1)). l-Ascorbyl 6-palmitate exhibited a reactivity similar to that of delta-tocopherol in homogeneous solution, but displayed a 1 order of magnitude lower fluorescence quenching efficiency in liposomes than the vitamin E constituents. Temperature effects on the laterally diffusion-controlled fluorescence quenching were large, with activation energies of 44 +/- 6 kJ mol(-1). The addition of cholesterol (0-30%) to POPC liposomes resulted only in slightly reduced diffusion coefficients. The combined results demonstrate that Fluorazophore-L can provide important physicochemical parameters for the understanding of antioxidant activity in biological environments.

Chiral resolution through precipitation of diastereomeric capsules in the form of 2:1 beta-cyclodextrin-guest complexes.

Preferential precipitation of one enantiomer from a racemic mixture of a camphanate ester of 2,3-diazabicyclo[2.2.2]oct-2-ene was induced by the formation of diasteromeric 2:1 beta-cyclodextrin-guest complexes. The precipitate was enriched with the (-)-enantiomer and the supernatant solution with the (+)-form of a camphanate ester, which was quantitatively analyzed in terms of differential binding constants and intrinsic solubilities of the 2:1 complexes. The enantiomeric excess in the precipitate was determined as 30 +/- 3% by induced circular dichroism.

Induced circular dichroism and structural assignment of the cyclodextrin inclusion complexes of bicyclic azoalkanes.

The stoichiometries and binding constants of the host-guest complexes between the bicyclic azoalkanes 1-6 and alpha-, beta-, and gamma-cyclodextrins (CDs) and the induced circular dichroism (ICD) of the complexes were analyzed. Assisted by proximity relationships obtained from 2D ROESY NMR spectra, the signs and intensities of the ICD spectra are interpreted in terms of the solution structures (co-conformations) of the CD complexes. The ICD assignments are based on the orientation-intensity ICD rules of Harata and Kodaka, which relate the ICD signs and intensities to the relative orientation of the electric dipole transition moment of the n,pi azo chromophore to the CD axis. The influence of the size of the guest and the host is discussed and the effect of introducing an additional chromophore (either a phenyl or a second azo group) on the ICD spectra is demonstrated.