A reusable QCR aptasensor for the detection of Brevetoxin-2 in shellfish.

Brevetoxins (BTX) are pharmacologically active, lipid soluble cyclic polyether neurotoxins that are known to cause a wide range of neurological symptoms in humans.Harvesting and consumption of infected molluscs provide an entry point for BTXs into, the food chain, causing long-term health effects on accumulation for individuals, commonly in people with a compromised immune system and existing allergies. This study is an acoustic assay that has been constructed using a 9 MHz AT-cut quartz crystal resonator modified by attaching a specific single-stranded DNA aptamer. The DNA oligo modifies its conformation to attach itself to the binding site of the incoming BTX molecule resulting in a change in frequency on the QCR. A small Δf value was observed for lower concentrations of BTX indicating a small change in mass deposited on the crystal surface, while the opposite was true for higher concentrations. Cross-species behavior was evaluated using samples of similar origin, molecular weight and a combination of two toxins. The LOD of the fabricated QCR is 220 nM which is lower than the maximum recommended residue limit in food samples. Fresh mussel samples were spiked with known concentrations of BTX to evaluate its sensitivity in a food matrix. No interaction with other compounds was observed. Overall, this sensor finds potential application in the food sector (fishing units) where mussels are tested and graded for allergens and toxins before reaching the customer.

Simplex optimization of acoustic assay for plasminogen activators.

This article discusses the optimization of a newly developed method for measuring the activity of plasminogen activators using a thickness-shear-mode acoustic sensor. A variable-size simplex algorithm was used for optimization. Preliminary tests were performed to design the first simplex. A desirability function was defined to translate each performance value to a membership value of 0 to 1. If there was more than one performance variable, their membership values were translated to an aggregated membership value using another function that considers their individual influence on sensor performance. Two rounds of optimization were carried out for streptokinase followed by a single optimization for tissue-type plasminogen activator. In the last optimization, ratios of control variables were used in order to reduce the number of parameters and to formulate easily adjustable assay conditions. The results showed the usefulness of the simplex method for optimizing this type of assay, and the importance of preliminary tests and prior knowledge in providing rapid convergence using fewer experiments. The optimized plasminogen activator assay can be considered a reference method for measurement of all members of this drug class.

Acoustic determination of performance and equivalence of plasminogen activators.

A reliable method for the measurement of different plasminogen activators is of great interest for both manufacturing and clinical medicine. A one-step assay based on a thickness shear mode acoustic sensor has been developed for this purpose. Two separate mixtures of substrates (fibrinogen and plasminogen) and enzymes (thrombin and the plasminogen activator) were mixed, and placed on the acoustic sensor surface. During the assay, the resonant frequency of a quartz crystal oscillating in the thickness shear mode was measured and used to find a characteristic clot dissolution time, from the sample addition to the time at the maximum dissolution rate. Calibrations of the acoustic assay were done for tissue-type plasminogen activator (t-PA) as well as for the other plasminogen activators: urokinase (u-PA); streptokinase (SK) and staphylokinase (SAK). All gave relative standard deviations of about 12%. Since the same method was used for all of the activators, their activities were compared, resolving the differences between their unit definitions. Linear relationships were found between urokinase and streptokinase which activate plasminogen directly and between t-PA and staphylokinase which require fibrin as a cofactor. The relationship between the groups was found to curve, indicating the difference between the two mechanisms. The acoustic method, therefore, may be used as a rapid and cost-effective reference method for the standardization and comparison of different plasminogen activators.

One-step thickness shear mode acoustic assay for plasminogen activators.

A new procedure is presented for the measurement of plasminogen activators using a thickness shear mode sensor and a modified version of the fibrin plate assay at the micro-scale. Separate, well-mixed solutions of the substrates fibrinogen and plasminogen, and enzymes thrombin and the plasminogen activator sample were mixed together and placed on the sensor surface. The temperature and evaporation were controlled during the assay. The clot dissolution time correlated well with the quantity of the plasminogen activator in the sample. The average relative standard deviation was 12.5%.

Acoustic coupling of transverse waves as a mechanism for the label-free detection of protein-small molecule interactions.

An on-line acoustic transverse wave device has been used to study the binding interactions of human serum albumin with the small molecule drug, warfarin. Four linking systems for the covalent attachment of the protein to the surface of the gold electrode of the sensor were employed, namely thioctic acid, cysteamine, an N-hydroxysuccinimide ester and 11-mercaptoundecanoic acid. All the attachment protocols involve the ability of thiols to form gold-sulfur bonds at the metal surface. The functional group present at the distal end of each thiol was chemically activated in order to facilitate covalent attachment of the protein. On-line sensor measurements of acoustic parameters show that the binding of warfarin to the protein can be detected, and depending on the linking monolayer used three of four possible combinations of changes in series resonance frequency and motional resistance are observed. Calculations of possible mass and thickness viscoelastic effects demonstrate that these conventional notions are invalid in terms of an explanation of the acoustic signals observed for the warfarin-protein interaction. The responses are ascribed to acoustic coupling phenomena.