A thermoelectrically stabilized aluminium acoustic trap combined with attenuated total reflection infrared spectroscopy for detection of Escherichia coli in water.

Acoustic trapping is a non-contact particle manipulation method that holds great potential for performing automated assays. We demonstrate an aluminium acoustic trap in combination with attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) for detection of E. coli in water. The thermal conductivity of aluminium was exploited to thermo-electrically heat and hold the acoustic trap at the desired assay temperature of 37 °C. Systematic characterisation and optimisation of the acoustic trap allowed high flow rates while maintaining high acoustic trapping performance. The ATR element serves not only as a reflector for ultrasound standing wave generation but also as a sensing interface. The enzyme conversion induced by alkaline phosphatase-labelled bacteria was directly monitored in the acoustic trap using ATR-FTIR spectroscopy. Sequential injection analysis allowed automated liquid handling, including non-contact bacteria retention, washing and enzyme-substrate exchange within the acoustic trap. The presented method was able to detect E. coli concentrations as low as 1.95 × 106 bacteria per mL in 197 min. The demonstrated ultrasound assisted assay paves the way to fully automated bacteria detection devices based on acoustic trapping combined with ATR-FTIR spectroscopy.

In-Line Ultrasound-Enhanced Raman Spectroscopy Allows for Highly Sensitive Analysis with Improved Selectivity in Suspensions.

Raman spectroscopy is a nondestructive characterization method offering chemical-specific information. However, the cross-section of inelastically (Raman) scattered light is very low compared to elastically (Rayleigh) scattered light, resulting in weak signal intensities in Raman spectroscopy. Despite providing crucial information in off-line measurements, it usually is not sensitive enough for efficient, in-line process control in conjunction with low particle concentrations. To overcome this limitation, two custom-made 1.4404 stainless-steel prototype add-ons were developed for in-line Raman probes that enable ultrasound particle manipulation and thus concentration of particles in suspensions in the focus of the Raman excitation laser. Depending on size and density differences between particles and the carrier medium, particles are typically caught in the nodal planes of a quasi-standing wave field formed in an acoustic resonator in front of the sensor. Two arrangements were realized with regard to the propagation direction of the ultrasonic wave relative to the propagation direction of the laser. The parallel arrangement improved the limit of detection (LOD) by a factor of ≈30. In addition to increased sensitivity, the perpendicular arrangement offers increased selectivity: modifying the frequency of the ultrasonic wave field allows the liquid or solid phase to be moved into the focus of the Raman laser. The combination of in-line Raman spectroscopy with ultrasound particle manipulation holds promise to push the limits of conventional Raman spectroscopy, hence broadening its field of application to areas where previously Raman spectroscopy has not had sufficient sensitivity for accurate, in-line detection.

An Acoustic Trap for Bead Injection Attenuated Total Reflection Infrared Spectroscopy.

In this work, we introduce a system combining an acoustic trap for bead injection with attenuated total reflection (ATR) infrared (IR) spectroscopy. By mounting an acoustofluidic cell hosting an ultrasound source on top of a custom-built ATR fixture we were able to trap beads labeled with the enzyme alkaline phosphatase without requiring any mechanical retention elements. Sequential injection analysis was employed for reproducible sample handling and bead injection into the acoustic trap. To showcase potential applications of the presented setup for kinetic studies, we monitored the conversion of p-nitrophenylphosphate into p-nitrophenol and phosphate via beads carrying the immobilized enzyme using ATR-IR spectroscopy. Retaining the labeled beads via ultrasound particle manipulation resulted in excellent experimental reproducibility (relative standard deviation, 3.91%). It was demonstrated that trapped beads remained stably restrained with up to eight cell volumes of liquid passing through the acoustofluidic cell. Beads could be discarded in a straightforward manner by switching off the ultrasound, in contrast to systems containing mechanical retention elements, which require backflushing. Multiple experiments were performed by employing different substrate concentrations with the same batch of trapped beads as well as varying the amount of enzyme present in the cell, enabling enzyme kinetic studies and emphasizing the application of the proposed setup in studies where enzymatic reuse is desired. This proves the potential of the acoustic trap combined with ATR-IR spectroscopy to monitor the activity of immobilized enzymes and its ability to perform complex bead-based assays.

Ultrasound-enhanced attenuated total reflection mid-infrared spectroscopy in-line probe: acquisition of cell spectra in a bioreactor.

This article presents a novel method for selective acquisition of Fourier transform infrared (FT-IR) spectra of microorganisms in-line during fermentation, using Saccharomyces cerevisiae as an example. The position of the cells relative to the sensitive region of the attenuated total reflection (ATR) FT-IR probe was controlled by combing a commercially available ATR in-line probe with contact-free, gentle particle manipulation by ultrasonic standing waves. A prototype probe was successfully constructed, assembled, and tested in-line during fed-batch fermentations of S. cerevisiae. Control over the position of the cells was achieved by tuning the ultrasound frequency: 2.41 MHz was used for acquisition of spectra of the cells (pushing frequency f(p)) and 1.87 MHz, for retracting the cells from the ATR element, therefore allowing spectra of the medium to be acquired. Accumulation of storage carbohydrates (trehalose and glycogen) inside the cells was induced by a lack of a nitrogen source in the feed medium. These changes in biochemical composition were visible in the spectra of the cells recorded in-line during the application of f(p) and could be verified by reference spectra of dried cell samples recorded off-line with a FT-IR microscope. Comparison of the cell spectra with spectra of trehalose, glycogen, glucose, and mannan, i.e., the major carbohydrates present in S. cerevisiae, and principal components analysis revealed that the changes observed in the cell spectra correlated well with the bands specific for trehalose and glycogen. This proves the applicability and capability of ultrasound-enhanced in-line ATR mid-IR spectroscopy as a real-time PAT method for the in situ monitoring of cellular biochemistry during fermentation.

Ultrasonic manipulation of yeast cells in suspension for absorption spectroscopy with an immersible mid-infrared fiberoptic probe.

Recent advances in combining ultrasonic particle manipulation with attenuated total reflection infrared spectroscopy of yeast suspensions are presented. Infrared spectroscopy provides highly specific molecular information about the sample. It has not been applicable to in-line monitoring of cells during fermentation, however, because positioning cells in the micron-thin measurement region of the attenuated total reflection probe was not possible. Ultrasonic radiation forces exerted on suspended particles by an ultrasonic standing wave can result in the buildup of agglomerates in the nodal planes, hence enabling the manipulation of suspended cells on the microscopic scale. When a chamber setup and a prototype in-line applicable probe were used, successful control over the position of the yeast cells relative to the attenuated total reflection sensor surface could be proven. Both rate of increase and maximum mid-infrared absorption of yeast-specific bands during application of a pushing frequency (chamber setup: 1.863 MHz, in-line probe: 1.990 MHz) were found to correlate with yeast cell concentration.

Acoustofluidics 22: multi-wavelength resonators, applications and considerations.

One important niche for multi-wavelength resonators is the filtration of suspensions containing very high particle concentration. For some applications, multi-wavelength ultrasound enhanced sedimentation filters are second only to the centrifuge in efficiency but, unlike the centrifuge they are easily adapted for continuous flow. Multi-wavelength resonators are also an obvious consideration when half-wavelength chambers are too small for a specific application. Unfortunately the formula, bigger = higher-throughput, does not scale linearly. Here we describe the relationships between chamber size and throughput for acoustic, electrical, flow and thermal convection actions, allowing the user to define initial parameters for their specific applications with some confidence. We start with a review of some of the many forms of multi-wavelength particle manipulation systems.

Acoustofluidics 21: ultrasound-enhanced immunoassays and particle sensors.

In part 21 of the tutorial series "Acoustofluidics--exploiting ultrasonic standing wave forces and acoustic streaming in microfluidic systems for cell and particle manipulation", we review applications of ultrasonic standing waves used for enhancing immunoassays and particle sensors. The paper covers ultrasonic enhancement of bead-based immuno-agglutination assays, bead-based immuno-fluorescence assays, vibrational spectroscopy sensors and cell deposition on a sensor surface.

Observation of particles manipulated by ultrasound in close proximity to a cone-shaped infrared spectroscopy probe.

The presented investigations aimed to enhance surface sensitive infrared spectroscopy for chemical analysis by ultrasonic particle manipulation. The combination of these techniques has the potential for new measurement concepts for use in the chemical analysis of suspensions. Local increases of particle concentration brought about by ultrasound could facilitate measurements of molecular-specific infrared spectra of the suspending phase and particles independently. By changing the frequency of an ultrasonic standing wave around 2 MHz it was possible to control the position of particles in respect to the optically sensitive region of the infrared spectroscope. Results obtained with a set-up that enabled us to explore the application of an ultrasonic standing wave to push suspended particles at or into mum distances of the sensing element of an in-line fiber optic probe and subsequently retract them from there are presented. Light micrographs suggested, that the task was successfully accomplished with polystyrene beads suspended in methanol, aggregates were manipulated to and from the cut surface of the truncated, cone-shaped fibre probe tip by changes of the ultrasonic frequency between 1.85 and 1.87 MHz. Feasibility was confirmed by infrared absorption spectra recorded when PTFE particles suspended in tetrahydrofuran were used.

Ultrasonic trapping of microparticles in suspension and reaction monitoring using Raman microspectroscopy.

An ultrasonic standing wave around 2 MHz has been used for trapping and concentration of suspended micrometer-size particles in a flow cell, whereas Raman microspectroscopy was used as a nondestructive technique to provide molecular information about the trapped particles. With this approach, detection and discrimination of different polymer microparticles based on their characteristic Raman spectra was performed. Dextran, poly(vinyl alcohol), and melamine resin-based beads, with and without functionalization, were used for this purpose. Furthermore, taking advantage of the flow-through characteristics of the cell and the versatility of the employed flow system, full control over the media surrounding the trapped particles was achieved. This allowed us to perform chemical reactions on the trapped particles and to monitor spectral changes in real time. Here retention of cation-exchanger beads loaded with silver ions and subsequent reduction of the silver ions was demonstrated. In this way, surface-enhanced Raman (SER) active beads were prepared and retained in the focus of the Raman microscope by means of the ultrasonic field. Injection of analytes in the flow system thus allowed recording of their SER spectra. Using 9-aminoacridine, a linear dependence of the found SER signal in the range from 1 to 10 microM has been achieved. The repeatability in the recorded SER intensities was on the order of 4-5%. This included bead retention, surface-enhanced Raman layer synthesis, and analyte detection.

A new immobilisation method to arrange particles in a gel matrix by ultrasound standing waves.

Ultrasonic forces may be used to manipulate particles in suspension. For example, a standing wave ultrasound (US) field applied to a suspension moves the particles toward areas of minimal acoustic pressure, where they are orderly retained creating a predictable heterogeneous distribution. This principle of ultrasonic retention of particles or cells has been applied in numerous biotechnological applications, such as mammalian cell filtering and red blood cell sedimentation. Here, a new US-based cell immobilisation technique is described that allows manipulation and positioning of cells/particles within various nontoxic gel matrices before polymerisation. Specifically, gel immobilisation was used to directly demonstrate that the viability of yeast cells arranged by an US standing wave is maintained up to 4 days after treatment. The versatility of this immobilisation method was validated using a wide range of acoustic devices. Finally, the potential biotechnological advantages of this US-controlled particle positioning method combined with gel immobilisation/encapsulation technology are discussed.