High Fundamental Frequency (HFF) Monolithic Quartz Crystal Microbalance with Dissipation Array for the Simultaneous Detection of Pesticides and Antibiotics in Complex Food.

As in the case of the food industry in general, there is a global concern about safety and quality in complex food matrices, such as honey, which is driving the demand for fast, sensitive and affordable analytical techniques across the honey-packaging industry. Although excellent techniques such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) are available, these are located in centralized laboratories and are still lacking in speed, simplicity and cost-effectiveness. Here, a new approach is presented where a competitive immunoassay is combined with a novel High Fundamental Frequency Quartz Crystal Microbalance with Dissipation (HFF-QCMD) array biosensor for the simultaneous detection of antibiotics and pesticides in honey. Concretely, thiabendazole and sulfathiazole residues were monitored in spiked honey samples. Results revealed that HFF-QCMD arrays provide a complementary and reliable tool to LC-MS/MS for the analysis of contaminants in these kinds of complex matrices, while avoiding elaborate sample pre-treatment. The good sensitivity achieved (I50 values in the 70-720 µg/kg range) and the short analysis time (60 min for 24 individual assays), together with the ability for multiple analyte detection (24 sensor array) and its cost-effectiveness, pave the way for the implementation of a fast on-line, in situ routine control of potentially hazardous chemical residues in honey.

Acoustic Array Biochip Combined with Allele-Specific PCR for Multiple Cancer Mutation Analysis in Tissue and Liquid Biopsy.

Regular screening of point mutations is of importance to cancer management and treatment selection. Although techniques like next-generation sequencing and digital polymerase chain reaction (PCR) are available, these are lacking in speed, simplicity, and cost-effectiveness. The development of alternative methods that can detect the extremely low concentrations of the target mutation in a fast and cost-effective way presents an analytical and technological challenge. Here, an approach is presented where for the first time an allele-specific PCR (AS-PCR) is combined with a newly developed high fundamental frequency quartz crystal microbalance array as biosensor for the amplification and detection, respectively, of cancer point mutations. Increased sensitivity, compared to fluorescence detection of the AS-PCR amplicons, is achieved through energy dissipation measurement of acoustically "lossy" liposomes binding to surface-anchored dsDNA targets. The method, applied to the screening of BRAF V600E and KRAS G12D mutations in spiked-in samples, was shown to be able to detect 1 mutant copy of genomic DNA in an excess of 104 wild-type molecules, that is, with a mutant allele frequency (MAF) of 0.01%. Moreover, validation of tissue and plasma samples obtained from melanoma, colorectal, and lung cancer patients showed excellent agreement with Sanger sequencing and ddPCR; remarkably, the efficiency of this AS-PCR/acoustic methodology to detect mutations in real samples was demonstrated to be below 1% MAF. The combined high sensitivity and technology-readiness level of the methodology, together with the ability for multiple sample analysis (24 array biochip), cost-effectiveness, and compatibility with routine workflow, make this approach a promising tool for implementation in clinical oncology labs for tissue and liquid biopsy.

A Real-Time Method for Improving Stability of Monolithic Quartz Crystal Microbalance Operating under Harsh Environmental Conditions.

Monolithic quartz crystal microbalance (MQCM) has recently emerged as a very promising technology suitable for biosensing applications. These devices consist of an array of miniaturized QCM sensors integrated within the same quartz substrate capable of detecting multiple target analytes simultaneously. Their relevant benefits include high throughput, low cost per sensor unit, low sample/reagent consumption and fast sensing response. Despite the great potential of MQCM, unwanted environmental factors (e.g., temperature, humidity, vibrations, or pressure) and perturbations intrinsic to the sensor setup (e.g., mechanical stress exerted by the measurement cell or electronic noise of the characterization system) can affect sensor stability, masking the signal of interest and degrading the limit of detection (LoD). Here, we present a method based on the discrete wavelet transform (DWT) to improve the stability of the resonance frequency and dissipation signals in real time. The method takes advantage of the similarity among the noise patterns of the resonators integrated in an MQCM device to mitigate disturbing factors that impact on sensor response. Performance of the method is validated by studying the adsorption of proteins (neutravidin and biotinylated albumin) under external controlled factors (temperature and pressure/flow rate) that simulate unwanted disturbances.

A Multichannel Microfluidic Sensing Cartridge for Bioanalytical Applications of Monolithic Quartz Crystal Microbalance.

Integrating acoustic wave sensors into lab-on-a-chip (LoC) devices is a well-known challenge. We address this challenge by designing a microfluidic device housing a monolithic array of 24 high-fundamental frequency quartz crystal microbalance with dissipation (HFF-QCMD) sensors. The device features six 6-µL channels of four sensors each for low-volume parallel measurements, a sealing mechanism that provides appropriate pressure control while assuring liquid confinement and maintaining good stability, and provides a mechanical, electrical, and thermal interface with the characterization electronics. We validate the device by measuring the response of the HFF-QCMD sensors to the air-to-liquid transition, for which the robust Kanazawa-Gordon-Mason theory exists, and then by studying the adsorption of model bioanalytes (neutravidin and biotinylated albumin). With these experiments, we show how the effects of the protein-surface interactions propagate within adsorbed protein multilayers, offering essentially new insight into the design of affinity-based bioanalytical sensors.

Detection of DDT and carbaryl pesticides in honey by means of immunosensors based on high fundamental frequency quartz crystal microbalance (HFF-QCM).

BACKGROUND: In recent years there has been a concern about the presence of pesticides in honey because residues of DDT and carbaryl were found in honey samples. Traditional techniques, such as chromatography, reach the required limits of detection (LOD) but are not suitable for in situ implementation in the honey-packaging industry due to their high cost and the need for highly qualified staff for routine operation. Biosensors offer simplicity, low cost, and easy handling for analytical purposes in food applications. RESULTS: Piezoelectric immunosensors based on high fundamental frequency quartz crystal microbalance (HFF-QCM) have been developed for the detection of carbaryl and DDT in honey. Biorecognition was based on competitive immunoassays in the conjugate-coated format, using monoclonal antibodies as specific immunoreagents. The assay LODs attained by the HFF-QCM immunosensors were 0.05 µg L-1 for carbaryl and 0.24 µg L-1 for DDT, reaching a similar level of detectability to that of the usual reference techniques. The practical LODs in honey samples were 8 µg kg-1 for carbaryl and 24 µg kg-1 for DDT. The immunosensors' analytical performance allow the detection of these pesticides in honey at EU regulatory levels with good accuracy (recovery percentages ranging from 94% to 130% within the working range of each pesticide standard curve) and precision (coefficients of variation in the 9-36% range). CONCLUSION: The proposed immunosensor is a promising analytical tool that could be implemented for quality control in the honey packaging industry, to simplify and to reduce the cost of the routine pesticide analysis in this appreciated natural food. © 2020 Society of Chemical Industry.

An ultrafast quartz crystal microbalance based on a frequency comb approach delivers sub-millisecond time resolution.

Quartz crystal microbalance with dissipation monitoring (QCMD) is a simple and versatile sensing technique with applications in a wide variety of academic and industrial fields, most notably electrochemistry, biophysics, quality control, and environmental monitoring. QCMD is limited by a relatively poor time resolution, which is of the order of seconds with conventional instrument designs at the noise level usually required. In this work, we present a design of an ultrafast QCMD with submillisecond time resolution. It is based on a frequency comb approach applied to a high-fundamental-frequency (HFF) resonator through a multifrequency lock-in amplifier. The combination allows us to reach data acquisition rates >10 kHz. We illustrate the method using a toy model of a glass sphere dropped on the resonator surfaces, bare or coated with liposomes, in liquid. We discuss some interesting features of the results obtained with the dropped spheres, such as bending of the HFF resonators due to the impact, sphere bouncing (or the absence of it), and contact aging.

Design and Validation of a 150 MHz HFFQCM Sensor for Bio-Sensing Applications.

Acoustic wave resonators have become suitable devices for a broad range of sensing applications due to their sensitivity, low cost, and integration capability, which are all factors that meet the requirements for the resonators to be used as sensing elements for portable point of care (PoC) platforms. In this work, the design, characterization, and validation of a 150 MHz high fundamental frequency quartz crystal microbalance (HFF-QCM) sensor for bio-sensing applications are introduced. Finite element method (FEM) simulations of the proposed design are in good agreement with the electrical characterization of the manufactured resonators. The sensor is also validated for bio-sensing applications. For this purpose, a specific sensor cell was designed and manufactured that addresses the critical requirements associated with this type of sensor and application. Due to the small sensing area and the sensor's fragility, these requirements include a low-volume flow chamber in the nanoliter range, and a system approach that provides the appropriate pressure control for assuring liquid confinement while maintaining the integrity of the sensor with a good base line stability and easy sensor replacement. The sensor characteristics make it suitable for consideration as the elemental part of a sensor matrix in a multichannel platform for point of care applications.

A High Fundamental Frequency (HFF)-based QCM Immunosensor for Tuberculosis Detection.

BACKGROUND: Tuberculosis, one of the oldest diseases affecting human beings, is still considered as a world public health problem by the World Health Organization. METHOD & MATERIAL: Therefore, there is a need for new and more powerful analytical methods for early illness diagnosis. With this idea in mind, the development of a High Fundamental Frequency (HFF) piezoelectric immunosensor for the sensitive detection of tuberculosis was undertaken. A 38 kDa protein secreted by Mycobacterium tuberculosis was first selected as the target biomarker. Then, specific monoclonal antibodies (MAbs) were obtained. Myc-31 MAb, which showed the highest affinity to the analyte, was employed to set up a reference enzyme-linked immunosorbent assay (ELISA) with a limit of detection of 14 ng mL-1 of 38 kDa antigen. RESULTS & DISCUSSION: For the development of the HFF piezoelectric immunosensor, 100 MHz quartz crystals were used as transducer elements. The gold electrode surface was functionalized by covalent immobilization of the target biomarker through mixed self-assembled monolayers (mSAM) of carboxylic alkane thiols. A competitive immunoassay based on Myc-31 MAb was integrated with the transducer as sensing bio-recognition event. Reliable assay signals were obtained using low concentrations of antigen for functionalization and MAb for the competitive immunoassay. Under optimized conditions, the HFF immunosensor calibration curve for 38 kDa determination showed a limit of detection as low as 11 ng mL-1 of the biomarker. The high detectability attained by this immunosensor, in the picomolar range, makes it a promising tool for the easy, direct and sensitive detection of the tuberculosis biomarker in biological fluids such as sputum.

Oxidative lung injury correlates with one-lung ventilation time during pulmonary lobectomy: a study of exhaled breath condensate and blood.

OBJECTIVES: During lung lobectomy, the operated lung is collapsed and hypoperfused; oxygen deprivation is accompanied by reactive hypoxic pulmonary vasoconstriction. After lung lobectomy, ischaemia present in the collapsed state is followed by expansion-reperfusion and lung injury attributed to the production of reactive oxygen species. The primary objective of this study was to investigate the time course of several markers of oxidative stress simultaneously in exhaled breath condensate and blood and to determine the relationship between oxidative stress and one-lung ventilation time in patients undergoing lung lobectomy. METHODS: This single-centre, observational, prospective study included 28 patients with non-small-cell lung cancer who underwent lung lobectomy. We measured the levels of hydrogen peroxide, 8-iso-PGF2α, nitrites plus nitrates and pH in exhaled breath condensate (n = 25). The levels of 8-iso-PGF2α and nitrites plus nitrates were also measured in blood (n = 28). Blood samples and exhaled breath condensate samples were collected from all patients at five time points: preoperatively; during one-lung ventilation, immediately before resuming two-lung ventilation; immediately after resuming two-lung ventilation; 60 min after resuming two-lung ventilation and 180 min after resuming two-lung ventilation. RESULTS: Both exhaled breath condensate and blood exhibited significant and simultaneous increases in oxidative-stress markers immediately before two-lung ventilation was resumed. However, all these values underwent larger increases immediately after resuming two-lung ventilation. In both exhaled breath condensate and blood, marker levels significantly and directly correlated with the duration of one-lung ventilation immediately before resuming two-lung ventilation and immediately after resuming two-lung ventilation. Although pH significantly decreased in exhaled breath condensate immediately after resuming two-lung ventilation, these pH values were inversely correlated with the duration of one-lung ventilation. CONCLUSIONS: During lung lobectomy, the operated lung is collapsed and oxidative injury occurs, with the levels of markers of oxidative stress increasing simultaneously in exhaled breath condensate and blood during one-lung ventilation. These increases were larger after resuming two-lung ventilation. Increases immediately before resuming two-lung ventilation and immediately after resuming two-lung ventilation were directly correlated with the duration of one-lung ventilation.

High-frequency phase shift measurement greatly enhances the sensitivity of QCM immunosensors.

In spite of being widely used for in liquid biosensing applications, sensitivity improvement of conventional (5-20MHz) quartz crystal microbalance (QCM) sensors remains an unsolved challenging task. With the help of a new electronic characterization approach based on phase change measurements at a constant fixed frequency, a highly sensitive and versatile high fundamental frequency (HFF) QCM immunosensor has successfully been developed and tested for its use in pesticide (carbaryl and thiabendazole) analysis. The analytical performance of several immunosensors was compared in competitive immunoassays taking carbaryl insecticide as the model analyte. The highest sensitivity was exhibited by the 100MHz HFF-QCM carbaryl immunosensor. When results were compared with those reported for 9MHz QCM, analytical parameters clearly showed an improvement of one order of magnitude for sensitivity (estimated as the I50 value) and two orders of magnitude for the limit of detection (LOD): 30µgl(-1) vs 0.66µgL(-1)I50 value and 11µgL(-1) vs 0.14µgL(-1) LOD, for 9 and 100MHz, respectively. For the fungicide thiabendazole, I50 value was roughly the same as that previously reported for SPR under the same biochemical conditions, whereas LOD improved by a factor of 2. The analytical performance achieved by high frequency QCM immunosensors surpassed those of conventional QCM and SPR, closely approaching the most sensitive ELISAs. The developed 100MHz QCM immunosensor strongly improves sensitivity in biosensing, and therefore can be considered as a very promising new analytical tool for in liquid applications where highly sensitive detection is required.

Love wave immunosensor for the detection of carbaryl pesticide.

A Love Wave (LW) immunosensor was developed for the detection of carbaryl pesticide. The experimental setup consisted on: a compact electronic characterization circuit based on phase and amplitude detection at constant frequency; an automated flow injection system; a thermal control unit; a custom-made flow-through cell; and Quartz /SiO2 LW sensors with a 40 µm wavelength and 120 MHz center frequency. The carbaryl detection was based on a competitive immunoassay format using LIB-CNH45 monoclonal antibody (MAb). Bovine Serum Albumin-CNH (BSA-CNH) carbaryl hapten-conjugate was covalently immobilized, via mercaptohexadecanoic acid self-assembled monolayer (SAM), onto the gold sensing area of the LW sensors. This immobilization allowed the reusability of the sensor for at least 70 assays without significant signal losses. The LW immunosensor showed a limit of detection (LOD) of 0.09 µg/L, a sensitivity of 0.31 µg/L and a linear working range of 0.14-1.63 µg/L. In comparison to other carbaryl immunosensors, the LW immunosensor achieved a high sensitivity and a low LOD. These features turn the LW immunosensor into a promising tool for applications that demand a high resolution, such as for the detection of pesticides in drinking water at European regulatory levels.

Validation of a phase-mass characterization concept and interface for acoustic biosensors.

Acoustic wave resonator techniques are widely used in in-liquid biochemical applications. The main challenges remaining are the improvement of sensitivity and limit of detection, as well as multianalysis capabilities and reliability. The sensitivity improvement issue has been addressed by increasing the sensor frequency, using different techniques such as high fundamental frequency quartz crystal microbalances (QCMs), surface generated acoustic waves (SGAWs) and film bulk acoustic resonators (FBARs). However, this sensitivity improvement has not been completely matched in terms of limit of detection. The decrease on frequency stability due to the increase of the phase noise, particularly in oscillators, has made it impossible to increase the resolution. A new concept of sensor characterization at constant frequency has been recently proposed based on the phase/mass sensitivity equation: Δφ/Δm ≈ -1/m(L), where m(L) is the liquid mass perturbed by the resonator. The validation of the new concept is presented in this article. An immunosensor application for the detection of a low molecular weight pollutant, the insecticide carbaryl, has been chosen as a validation model.

Clinical-therapeutic management of thoracoscopy in pleural effusion: a groundbreaking technique in the twenty-first century.

INTRODUCTION: The aim of this study was to investigate the effectiveness of thoracoscopy in the diagnosis of non-affiliated pleural effusions (PE). MATERIAL AND METHODS: A five-year prospective study including data from 110 patients that were clinically diagnosed as benign (14.5%), malign (34.5%) and non-affiliated (50.9%). PE in patents without oncology disease and negative biopsy or cytology were considered as benign. Malignant diagnosis was established according to a pleural biopsy, compatible cytology and/or clinical features. Remaining cases were considered as non-affiliated. Thoracoscopy was done under local anaesthesia and sedation. RESULTS: Thoracoscopy confirmed previous clinical diagnosis of benignity and malignity. Regarding non-affiliated patients, 30.35% were diagnosed after thoracoscopy as unspecific pleuritis, 17.86% mesothelioma and 1.79% pleural tuberculosis (TBC). The other 48.21% of patients reported as non-affiliated were diagnosed with pleural carcinoma. Statistical analysis did not reveal differences between frequencies analysed. CONCLUSIONS: Our results indicate that thoracoscopy is a cost-effective and reliable technique for obtaining histological diagnosis in PE and also allows a directed pleurodesis if indicated.

A Review of Interface Electronic Systems for AT-cut Quartz Crystal Microbalance Applications in Liquids.

From the first applications of AT-cut quartz crystals as sensors in solutionsmore than 20 years ago, the so-called quartz crystal microbalance (QCM) sensor isbecoming into a good alternative analytical method in a great deal of applications such asbiosensors, analysis of biomolecular interactions, study of bacterial adhesion at specificinterfaces, pathogen and microorganism detection, study of polymer film-biomolecule orcell-substrate interactions, immunosensors and an extensive use in fluids and polymercharacterization and electrochemical applications among others. The appropriateevaluation of this analytical method requires recognizing the different steps involved andto be conscious of their importance and limitations. The first step involved in a QCMsystem is the accurate and appropriate characterization of the sensor in relation to thespecific application. The use of the piezoelectric sensor in contact with solutions stronglyaffects its behavior and appropriate electronic interfaces must be used for an adequatesensor characterization. Systems based on different principles and techniques have beenimplemented during the last 25 years. The interface selection for the specific application isimportant and its limitations must be known to be conscious of its suitability, and foravoiding the possible error propagation in the interpretation of results. This article presentsa comprehensive overview of the different techniques used for AT-cut quartz crystalmicrobalance in in-solution applications, which are based on the following principles:network or impedance analyzers, decay methods, oscillators and lock-in techniques. Theelectronic interfaces based on oscillators and phase-locked techniques are treated in detail,with the description of different configurations, since these techniques are the most used inapplications for detection of analytes in solutions, and in those where a fast sensorresponse is necessary.

A contribution to solve the problem of coating properties extraction in quartz crystal microbalance applications.

The problem of coating properties extraction in quartz crystal resonator (QCR) applications is one of the challenging tasks of QCR applications, not completely solved even in theoretical conditions. The present work demonstrates that the problem is determined only for a set of three parameters of the coating, which concentrate the four coating properties. The following parameters are chosen: the surface mass density m8 = p1h1, the loss tangent tan delta1 = G''1/G'1, and the magnitude of the characteristic impedance /Z1(c)/ = (p1/G1/)(1/2). An algorithm is proposed that permits, in ideal conditions, an unambiguous extraction of these parameters, starting only from the admittance spectrum of a three-layer compound resonator, obtained from the one-dimensional transmission-line model (TLM) around the resonance. The algorithm introduces three additional improvements in relation to other routines: the calculation time is drastically reduced, the problem of erroneous solutions related to relative minima in typical fitting routines is eliminated, and a systematic error analysis in the extraction of the coating properties in real practice can be carried out. The use of the proposed algorithm as a tool for studying the effects of different phenomena such as slipping or surface roughness is introduced.

Combined thoracotomy and laminectomy for spinal cavernomas with intrathoracic growth.

Dumbbell shaped epidural cavernomas are uncommon lesions. Of these, the extraforaminal extension is usually limited, and the lesions are removed surgically by an extended laminectomy. We present two cases of dumbbell shaped epidural cavernomas with a considerable intrathoracic growth. They were resected through a combined thoracotomy and laminectomy. Only in the case in which a wide approach to the posterior mediastinum was performed it was possible to completely remove the lesion. Since it is possible to anticipate this histological diagnosis by the magnetic resonance imaging characteristics, we propose that a wide transthoracic approach should be performed associated to a laminectomy if a complete resection of the lesion is the goal.

Cristales Piezoeléctricos de Cuarzo en Aplicación como Microbalanza QCM / Quartz Crystals Piezoelectric in Application how Microbalance QCM

En las ultimas décadas se ha venido desarrollando el campo de los biosensores, adaptándolos para múltiples aplicaciones tales como: determinación de virus, bacterias, pesticidas, gases (CO2) y en general cualquier partícula contra la cual puedan ser diseñados anticuerpos específicos. En el presente artículo exponemos un trabajo con cristales de cuarzo, para los cuales buscábamos aplicaciones que pudieran ser llevadas a un campo comercial. Inicialmente se realizaron mediciones para determinar las concentraciones óptimas de entrecruzadores y anticuerpos para realizar la correcta modificación del cristal, que permitieran detectar partículas de carbaryl (sustancia utilizada en múltiples pesticidas). Luego se expone una revisión y recopilación de diferentes esquemas de inmovilización para unir moléculas a los electrodos de oro sobre la superficie del cristal y algunos procesos de regeneración del mismo. Por último se replantea el esquema inicialmente utilizado y se proponen dos nuevos esquemas para realizar su evaluación...