WO2024240708A1

WO2024240708A1 - Method and apparatus for the detection of biomolecules in a liquid sample

Info

Publication number: WO2024240708A1
Application number: PCT/EP2024/063835
Authority: WO
Inventors: Electra Gizeli; Angelos NTIMTSAS
Original assignee: Foundation for Research and Technology-Hellas
Current assignee: Foundation for Research and Technology-Hellas
Priority date: 2023-05-19
Filing date: 2024-05-18
Publication date: 2024-11-28
Anticipated expiration: 2025-11-19

Abstract

A method and an apparatus for the detection of a biomolecule in a liquid sample which involves the use of an acoustic sensor (6), a removable flow strip (7), which is positioned above the acoustic sensor (6) so that the flow strip (7) and the sensing area of the acoustic sensor (6) form a capillary space, means for injecting liquid sample onto the flow strip, and a detector configured to detect the signal of the acoustic sensor.

Description

METHOD AND APPARATUS FOR THE DETECTION OF BIOMOLECULES IN A LIQUID SAMPLE

Field of the invention

The present invention relates to an apparatus and a method for performing biomolecular detection assays using an acoustic sensor.

Background of the invention

The advent of new diagnostic protocols and methods oriented towards Point-of-Care (PoC) applications, enabled the development of portable and low-cost platforms that do not require specialized facilities and trained staff. Thus, it is nowadays possible for remote areas to have access in urgent healthcare examinations (e.g. HIV detection), for suppliers and authorities to perform agricultural and food safety monitoring of the available products/facilities, and for individuals to perform home testing (e.g. SARS-CoV-2 infection) (Choi et al., 2019). Most of the PoC diagnostics are used for the detection of proteins, metabolites/dissolved ions, and nucleic acid in complex samples such as blood, saliva, nasopharyngeal swab, and sweat (Wang et al., 2021). A recent gold standard for PoC testing is the lateral flow assay (LFA) that is used for the detection of hormones (e.g. pregnancy test), proteins (e.g. antibody test) and some DNA/RNA targets (Boehringer & O’Farrell, 2021). The detection method is based on the immobilization of a specific molecule on the surface of a supporting membrane that will act as an anchor point for the target molecule in the sample. The crude sample is usually prepared in a specific buffer that will perform lysis of the cells or viral particles. Upon lysis, the target will bind to complementary molecules, such as antibodies or DNA probes, labelled with fluorescent dyes or gold nanoparticles, and the conjugate/complex will flow through the surface of the membrane. The complex is further bound to the membrane on the immobilized molecules and as a result a colored line will appear in case that the target is present in the sample. Even though this approach is usually rapid and cost-effective, a major drawback is that the assay is qualitative with a yes or no result, setting a boundary on further analysis and treatment/handling/clinical importance, while the qualitative result (i.e. the color change) may subject the assay to user bias and therefore report false results. Furthermore, label free, quantitative and easily adjustable biosensing applications are preferable in cases with: target variability/uncertainty, target with quantitative importance and expensive labeling molecules.

Towards this direction, acoustic sensors have been developed in recent years for label free applications. Based on the propagating wave, acoustic sensors can be either a Bulk Acoustic Wave type device (e.g., Quartz Crystal Microbalance, Thickness Shear Mode resonator), where the frequency and the dissipation of the fundamental oscillation and its harmonics are monitored in real-time, while they can also be a Surface Acoustic Wave (SAW) type device supporting a shear horizontal type acoustic wave (Lange, 2019). In the case of SAW sensors, a high frequency oscillator excites a pair of interdigital transducers (IDTs) on the surface of a piezoelectric material (e.g. quartz, lithium niobate, lithium tantalate) and the electromagnetic wave is converted to a mechanical oscillation (shear wave) that propagates on the surface of the crystal and it may be confined closer to the surface with the use of a waveguide material (e.g. Love Wave device). The propagated wave passes through a sensing area, where the adsorption of an entity will affect directly the amplitude and the phase of the oscillation. Finally, the oscillation will meet a second pair of IDTs and the mechanical wave will be converted back to an electromagnetic wave due to the piezoelectric effect of the substrate (Fogel et al., 2016; Primiceri et al., 2018). SAW devices that employ IDTs to generate a shear wave are: Love Wave, Surface Skimming Bulk Wave, Acoustic Plate Mode, Bleustein-Gulyaev wave or Surface Transverse Wave, while there also devices that may be non-IDT based (electromagnetically excited shear acoustic wave) or the wave excitation takes place using a thin membrane (e.g., Flexural Plate Wave, Lamb wave device etc.). In principle, by comparing the input and output amplitude/phase changes, adsorption of molecules on a specifically modified surface can be monitored in real-time. Currently, acoustic wave platforms that are intended to detect the binding of molecules and perform quantitative analyses need typically a constant liquid flow on the surface of the sensor, which can be achieved with the use of microfluidic chambers and channels that come in contact with the surface of the sensor. However, this poses two major limitations; firstly, the microfluidic apparatus should be extensively optimized and engineered in order to minimize attenuation losses and leaks (Papadakis et al., 2017), thus increasing manufacturing cost, and secondly the placement of the microfluidic channels on the acoustic sensor can be a demanding task for an unexperienced user and it may result in reduced sensitivity and/or erroneous results (Zida et al., 2021). Finally, the high working frequency of the acoustic sensors, requires complex and sophisticated instrumentation, which is difficult to be integrated in a portable platform that will find applications in the point- of-need (Nguyen et al., 2017; Depold et al., 2021). Up to now, there is not a portable, user- friendly and low-cost acoustic wave platform employing microfluidics to overcome the previously mentioned limitations and capable to perform a variety of detection assays.

An essential feature that will allow also acoustic sensor platforms to find applications outside the research field, is the realization of a low-cost and simplified interface for introducing liquid samples, without attenuating the acoustic wave. An alternative approach for immunoassay experiments without flow has been proposed by tst biomedical electronics Co., Ltd. (Peng et al., 2021 ; Cheng et al., 2022), where SH-SAW sensors are used for the detection of specific target molecules, however the samples are directly injected on the surface of the sensor, which sets a limitation regarding any intermediate washing steps (for example washing with a buffer solution) or using more complex assays that require the injection of multiple reagents or biomolecules. Even though there is a plethora of microfluidic arrangements and well-established manufacturing approaches, the acoustic wave technology currently sets important limitations regarding the reusability of the apparatus, the disposal of the materials and the acoustic sensors after each experiment and the user experience and skills that are required to use the apparatus. For this reason, the current platforms are dedicated for research environments, and therefore, the potential applications of acoustic sensors in the medical and industry field, have not been exploited yet.

Summary of the invention

The present invention provides an apparatus for the detection of biomolecules in a liquid sample. According to the present invention, the liquid sample is introduced on the sensing area of an acoustic wave sensor, without significantly attenuating the propagating wave. Furthermore, with the present invention it is possible to perform nucleic acid and protein assays and reuse the apparatus for multiple tests. To achieve this, the inventors have developed a method and an apparatus to monitor the adsorption of biomolecules and obtain quantitative results in real-time. According to the present invention, a capillary space is formed between a flow strip and the sensing area of the acoustic sensor without the need for confinement of the liquid with side walls. This is achieved by placing a flow strip in close proximity to the acoustic sensor. The liquid sample flows towards the parallel plate arrangement and forms a capillary interface. In addition, the inventors have provided an apparatus for real-time monitoring and recording of amplitude and phase changes on an acoustic sensor. The apparatus may further include a sample preparation station and a thermally conductive element for heating and/or cooling the sample for performing nucleic acid amplification assays, such as isothermal nucleic acid amplification assays.

Brief description of the drawings

Figure 1 shows an apparatus according to the present invention, with a thermally conductive block, a heating/cooling element, and a sample preparation station.

Figure 2 shows a top view of a sensing module of an apparatus according to the present invention with a printed circuit board (PCB), two radio frequency (RF) switches, an acoustic sensor, a holder, and a flow strip.

Figure 3 shows a side view of a sensing module of an apparatus of the present invention comprising the strip and the acoustic sensor. The figure shows an acoustic sensor, a flow strip connected with an absorbent pad, an injection hollow needle and spacers for the flow strip positioning. A magnified view of the capillary interface between the acoustic sensor and the flow strip is presented in the upper part of Figure 3. Inside the capillary interface, the interaction between two biomolecules, for example a biotinylated molecule (circular molecules) and streptavidin (oval molecules) takes place.

Figure 4 shows a top view of a sensing module of an apparatus according to the present invention with a printed circuit board (PCB), two radio frequency (RF) switches, an acoustic sensor, a flow strip with its absorbent pad, and a surrounding enclosure.

Figure 5 shows a side view of an apparatus according to the present invention, with a lid and a heating/cooling element that may facilitate four different isothermal (or not) amplification assays and a housing unit that facilitates the capillary interface on an acoustic sensor. Also, a hollow tube and an injection needle on the center, for aligning and penetrating tubes that are intended to introduce samples on the acoustic sensor.

Figure 6 shows an example test of the acoustic response over different frequencies before the placement of the flow strip on top of the acoustic device (a) and after the placement of the flow strip at a specific height over the sensing area of the acoustic device (b). In both figures, the maximum value of amplitude is pinpointed.

Figure 7 shows an example test of the acoustic response over different frequencies under the formation of the capillary interface (a), and the real-time adsorption of DNA ladder (from 100 base pairs to 1517 base pairs) on a functionalized with PLL-g-PEG (Poly-L-Lysine grafted with Polyethylene glycol side chains) surface (b).

Figure 8 shows an example test of the acoustic response over different frequencies under the formation of the capillary interface (a), and the real-time adsorption of BSA (Bovine Serum Albumin) protein on a functionalized with PLL (Poly-L-Lysine) surface (b).

Detailed description of the invention

The present invention provides an apparatus for the detection of a biomolecule in a liquid sample, wherein the apparatus comprises an acoustic sensor, a removable flow strip, means configured to position the flow strip above the acoustic sensor so that the flow strip and the sensing area of the acoustic sensor form a top and a bottom parallel plate of a capillary space, wherein the capillary space does not comprise side plates and wherein the width of the flow strip is equal or smaller than the width of the sensing area of the acoustic sensor, means for injecting the liquid sample onto the flow strip, and a detector configured to detect the signal of the acoustic sensor.

The present invention also provides a process for the detection of a biomolecule in a liquid sample, wherein the process comprises the steps of a) injecting the liquid sample onto a flow strip, wherein the flow strip is removably positioned above the sensing area of an acoustic sensor so that the flow strip and the sensing area of the acoustic sensor form a top and a bottom parallel plate of a capillary space, wherein the capillary space does not comprise side plates and wherein the width of the flow strip is equal or smaller than the width of the sensing area of the acoustic sensor, and b) detecting the signal of the acoustic sensor.

Preferably, the acoustic sensor is a Bulk Acoustic Wave (BAW) type sensor, or a Surface Acoustic Wave (SAW) type sensor.

According to the present invention, the flow strip of the apparatus is removable. This means that the strip can be removed, for example after the completion of the assay. The flow strip should be made of a material that does not interfere with the acoustic wave signal. Moreover, it should be inert or resistant to the reagents and the molecules used in the detection method. For example, the flow strip can be made from metal, glass, plastic, organic or inorganic polymers, such as nitrocellulose, or even from the combination of the aforementioned materials. Preferably, the flow strip is made of backed nitrocellulose. More preferably, the flow strip is made of polymer-backed nitrocellulose. Even more preferably, the flow strip is made of polyester-backed nitrocellulose. The flow strip may, for example, be a membrane used for a lateral flow test.

The flow strip may be disposable or reusable. Preferably, the flow strip is disposable.

According to the present invention, the liquid sample is injected onto the flow strip. Preferably, the means for injecting the liquid sample are configured to inject a constant flow of the sample. The injection of a constant flow can be performed by using various means, well known in the art. For example, the liquid sample can be injected onto the flow strip by using a pump, such as a peristaltic pump or a precision pipette.

According to the present invention, the flow strip is so positioned to form parallel plates of a capillary space between the strip and the sensing area of the acoustic sensor. The sensing area of the acoustic sensor is the area between the IDTs where the detection of biomolecules takes place. By positioning the flow strip in close proximity to the surface of the acoustic sensor, the capillary action will force the liquid to travel across the interface between the flow strip and the acoustic sensor. The capillary effect can be formed only when there is a narrow space between the two surfaces. If the flow strip is in contact with the sensor, the capillary will collapse and the liquid will spread in unwanted areas of the apparatus.

The width of the flow strip is adjusted based on the width of the sensing area of the acoustic sensor. Thus, the width of the flow strip is equal or smaller than the width of the sensing area of the acoustic sensor.

The present inventors have surprisingly found that there is no need for placing side plates in the capillary space formed between the flow strip and the sensing area of the sensor in order to confine the liquid sample within the sensing area and to prevent leaks to the surrounding area. Thus, according to the present invention, the liquid moves along the longitudinal axis of the strip in the space between the flow strip and the acoustic sensor without been confined by side plates. The present inventors have also found that when the capillary space comprises side plates, significant losses of the acoustic wave are produced. Therefore, the present invention, in which the capillary space does not comprise side plates, provides the advantage of increased sensitivity and enhanced signal acquisition. Furthermore, the manufacture of the apparatus of the present invention is less complex and less expensive compared to the manufacture of apparatuses of the prior art that use capillary based systems.

Preferably, the surface of the acoustic sensor is coated with a waveguide layer, such as a polymer and/or a photoresist, that increases the sensitivity of the sensor.

The gap between the surface of the flow strip and the surface of the acoustic sensor may be adjusted based on the flow rate of the sample that is required for a certain assay.

Preferably, the flow rate of the liquid sample on the flow strip is from 2pL/min to 50 pL/min. More preferably, the flow rate is from 5 pL/min to 35 pL/min. Even more preferably, from lOpL/min up to 25pL/min.

Preferably, the gap between the surface of the flow strip and the surface of the acoustic sensor is from 0.1 mm to 0.5mm.

The creation of the capillary space between the flow strip and the sensing area of the acoustic sensor enables the flow of the sample along the sensing area of the sensor and the acquisition of the acoustic signal. Thus, the present invention does not involve the use of microfluidic chambers and/or channels that come in contact with the surface of the sensor, as suggested in the prior art. This means that the limitations associated with the use of microfluidic chambers and/or channels, such as the complexity and cost of production are significantly improved with the present invention. Furthermore, the present invention provides great versatility, since it can be used under various liquid flows by simply adjusting the gap between the flow strip and the acoustic sensor. In addition, the flow strip can be disposable, which means that after the completion of an assay the flow strip can be replaced by a new flow strip and the apparatus can be used in a new assay.

Preferably, the biomolecule is selected from the group consisting of a peptide, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a saccharide, an oligosaccharide, and a lipid.

Preferably, an absorbent pad is attached to the flow strip and is configured to collect the liquid from the flow strip downstream from the acoustic sensor. The absorbent pad is made of any material that may absorb a liquid, preferably water. For example, the absorbent pad may be made of paper, cotton or a polymer, such as cellulose. The absorbent pad equalizes the flow of the liquid that passes along the flow strip and retains the liquid wastes. The absorbent material should be able to retain the total volume that is required to perform the assay.

Preferably, the apparatus of the present invention further comprises a sample preparation station.

The apparatus of the present invention may also comprise a heat exchanger for increasing or reducing the temperature of the sample.

According to an embodiment of the present invention, the sensing area of the acoustic sensor is functionalized with a molecule/layer that can bind specifically to the biomolecule of the liquid sample. For example, the sensing area of the acoustic sensor can be functionalized with antibodies specific for a protein, or even antibodies conjugated with nanoparticles for acoustic signal enhancement. The same approach can also be used for detecting other molecules, like hormones, chemicals, ions, or even living/non-living entities like cells, microorganisms, and viruses.

The assay and the target depend on the need of the user/facility/laboratory and it can be either a protein, for example an antibody, or a genetic target, for example a DNA or RNA sequence.

For detecting nucleic acids, an amplification step may be needed for detecting the presence of the target in a sample with higher sensitivity. For the nucleic acid amplification assay a conventional method such as polymerase chain reaction (PCR) may be used for the amplification of a sequence, or modern, less time consuming and isothermal methods can be used for the amplification step. Some examples of the latter are: recombinase polymerase amplification (RPA), SSB-Helicase Assisted Rapid PCR (SHARP), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), reverse transcript (RT) variants of the aforementioned methods, and many more. Preferably, for detecting nucleic acids LAMP or RPA (with RT variants) amplification methods are used. The adsorption of the amplified products on the sensor surface may be done with non-specific methods (e.g., adsorption of DNA on positively charged polymers) or with specific methods (e.g., Biotin modified DNA molecules that bind on sensor coated with streptavidin).

Figures 1 to 5 show embodiments and aspects of an apparatus according to the present invention.

According to a preferred embodiment, the apparatus of the present invention further comprises a sample preparation station (1), such as that shown in Figure 1. In the sample preparation station (1), the sample may be combined in an assay vessel with reagents, which are used in the performance of a certain assay, such as buffers, peptides, polymers, antibodies, primers, complementary sequences and the like.

The apparatus of the present invention may further comprise a heat exchanger for increasing or reducing the temperature of the sample.

Preferably, the heat exchanger comprises an irradiating source (e.g. infrared light) that elevates the temperature of the sample at the desired level. More preferably, the heat exchanger comprises a resistive element that comes in contact with a thermal conductive material, such as copper or aluminum, which may be properly machined to receive the vessels (e.g. heating block). Even more preferably, as it is shown in Figure 1 , the heat exchanger comprises a Peltier module (2) that may be used to either transfer or absorb heat from a thermally conductive block (3) and increase/reduce the temperature of a sample, respectively. Preferably, the heat exchanger is also able to cool down and preserve samples at a low temperature. For example, the heat exchanger may be capable to reach high temperatures (e.g. 100°C) and low temperatures (e.g. 0°C). Preferably, the heat exchanger may transit between different temperatures in a short amount of time with precision (using for example a Proportional-lntegral-Derivative [PID] controller), thus enabling the use of multiple detection assays that may include lysis of cells/microorganisms/viruses and performing molecular amplification methods that require more than two different temperature levels, such an example is PCR.

The acoustic sensor may be placed horizontally on a flat surface. Preferably, the surface should be resistive to moisture and chemical reagents. Preferably, the surface is made from a polymer, glass, or metal (coated or not). Preferably, the sensor is mounted on a printed circuit board (PCB) with any of the aforementioned materials and/or protective coatings. With this arrangement, the same board may be used for mounting complementary circuitry and electrically connecting the device directly on the PCB.

Figure 2 shows the sensing module of an apparatus according to the present invention, such as the apparatus of Figure 1 or Figure 5. The module comprises a PCB (4) that has circuitry to support any sort of radio frequency (RF) switches (5), their complementary components (e.g. resistors, capacitors, connectors) and the acoustic sensor (6). The RF switches may be used to select between different sensing areas during the sample injection. The RF switches may also be used to switch between different channels and monitor all the available sensing areas of the acoustic sensor during the data acquisition. The RF switches are preferably capable for instant state change between the sensing areas, without attenuating or affecting by any sort the input and output signal to and from the acoustic sensor, respectively. In the embodiment of Figure 2, two general purpose surface mount package (SMD) (5) non-reflective switches are used, which offer high isolation between the channels and they also have low insertion losses (less than 0.6 dB at frequencies less than 1 GHz). Finally, the state of each switch may be controlled by a microcontroller automatically, or manually by a user.

For an effective detection of a target molecule, an acoustic sensor may be used to monitor the adsorption of an entity on the surface in real-time. The prior art methods and apparatus, suggest the use of microfluidic channels to introduce samples on acoustic sensors. The arrangement, the surface area and the materials that are used for the fabrication of microfluidic apparatus require extensive engineering, post-processing methods and technical oriented personnel to evaluate the efficiency of the final microfluidic structure. Even though there is a well-established microfabrication industry that can mass produce microfluidic apparatus, the aforementioned technical limitations and practical difficulties, such as placing a microfluidic gasket in the field by an unexperienced operator, hamper the reliable use of acoustic sensors as monitoring devices at the point-of-care. Still, the relatively high cost of a disposable microfluidic chip, the energy loss resulted from carefully sealing the flow cell on the acoustic sensor and the need to eliminate any bubbles produced inside the microfluidic channels, make this approach difficult to control and implement outside the lab. The present inventors have now surprisingly found that the use of microfluidic apparatus can be avoided and replaced effectively by creating a capillary interface on the surface of an acoustic sensor.

According to the present invention, the liquid sample flows along the sensing area of an acoustic sensor, by creating a capillary flow interface. The capillary effect is formed solely on the sensing area of the acoustic sensor.

Figures 2, 3, and 4 show different views of a sensing module of an apparatus according to the present invention, such as the apparatus of Figure 1 , or Figure 5, and of the positioning of the flow strip relative to the acoustic sensor. The flow strip (7) that, in this embodiment, is aligned vertically to the propagation of the acoustic wave, is positioned in a specific height from the surface of the acoustic sensor (6) and aligned with respect to the sensor surface in order to create the capillary space solely on the sensing area of the acoustic sensor. The flow strip (7) may be properly positioned and held in place by using a holder (8) made from a hydrophobic material that does not affect the acoustic signal, such as glass/plastic/polymer/metal. Preferably, the holder may comprise a hollow channel where the flow strip (7) will be placed, or may comprise an adhesive to hold the flow strip (7) in proper position. For example, the holder (8) can be made from a glass slide coated with a hydrophobic material, in order to prevent any capillary interface collapse due to overflow and/or strip misplacement.

Preferably, the distance/gap between the flow strip (7) and the sensor (6) is adjustable. The gap between the two surfaces may be adjusted with respect to the flow rate that is required for the assay. The holder height and subsequently the flow strip height from the sensor surface, may be adjusted by different means. For example, by using metal spacers (9) with predetermined dimensions and magnets (10) on the top surface of the holder (8), the height can be precisely adjusted based on the desired flow, while it allows easy and rapid access to change the flow strip. The gap between the two surfaces can be measured directly by using metric feelers gauges, or by subtracting the height of the flow strip surface to the back of the holder, from the total flow strip and holder height. The present inventors have found that by changing the flow strip height, the apparatus can support different flow rates, suitable for an acoustic experiment.

As shown in Figures 2, 3 and 4, the flow strip (7) is a paper-like strip that acts as a channel to guide the capillary along the acoustic sensor surface. Preferably, the flow strip (7) is made of backed nitrocellulose membrane, for example, a membrane used for a lateral flow test. The nitrocellulose membrane may be blocked (for example with Bovine Serum Albumin, BSA) to reduce nonspecific binding on its surface. Furthermore, the flow strip (7) may be joined in one end with a glass fiber pad (not shown in the Figures), where the liquid sample could be injected and filtered in case that the sample has unwanted particles and contaminants. Alternatively, the strip (7) may have a hydrophobic inlet, which comes in direct contact with a hollow needle (11) that is connected with an assay vessel (for example, through a peristaltic pump). The injection of the fluid on the capillary interface may be done by placing the flow strip (7) above the hollow needle (11) and the support structure (12) that secures the inlet of the flow strip (7) in proper position, while the surrounding space is coated with a hydrophobic material (13) in order to force the fluid towards the capillary space. On the other end of the strip (7), i.e. downstream of the acoustic sensor, an absorbent pad (14) is attached to the flow strip (7) in order to equalize the flow and retain liquid wastes. The flow rate may be adjusted to the desired level based on the flow strip height above the sensor surface in order to counterbalance any drift that may be present on the sensor signal and use an equilibrated flow of buffer solution (or water) on the surface as a reference state/signal. Upon introducing the diluted (in buffer solution) target molecules into the capillary space, the adsorption of the target entities on the sensor surface will take place and the acoustic signal will deviate from the previously mentioned reference state until reaching a new equilibrium. The difference between the two states can be used to characterize the sample either qualitatively and/or quantitatively. The waste liquid and molecules that are not adsorbed on the sensor surface, are collected by the absorbent pad. The absorbent material should be able to retain the total volume that is required to perform the assay. Preferably, for a flow rate of 15pL/min, and a gap of 0.5mm, an absorbent cotton pad with a length of 56 mm and width of 17 mm is capable to maintain up to 780pL of total test volume.

As presented in Figure 4, the PCB board (4) with the circuitry and the acoustic sensor (6), may be held in place within a supporting base (15). A top lid (16) may be used to enclose the structure. The base and the lid may be secured together by the means of neodymium magnets (17).

The sample under examination may be transferred to the inlet of a flow strip (7), by using, for example, a peristaltic pump (not shown in the Figures). As described earlier in relation to Figure 1 and presented in Figure 5, the sample that is prepared in the sample preparation station (1) may be transferred to an assay vessel that can be penetrated by a hollow needle (injection point) (18) and the sample is transferred to the flow strip (7) through a tubing with the use of a pump or any other suitable means. The tubing should be inert and resistant to the reagents that are used. A protective cap (19) is placed on top of the injection point (18). The tubing is preferably flexible for handling convenience. If a pump is used for transferring the sample, it may be programmable by means of a microcontroller and precisely adjustable regarding the flow rate. For example, a microcontroller with an adjustable pulse width modulated (PWM) output may be used to control a pump. Finally, the top lid (21), on which the bottom side can also be used as the sample preparation station (1), encloses and protects the apparatus and the sensing module with the acoustic sensor (6).

The detection of target molecules in a sample includes the monitoring of the acoustic wave properties during an assay. Specifically, binding of molecules on a functionalized surface of an acoustic sensor may be monitored in real-time by measuring the amplitude/gain and phase changes during a testing procedure. Prior art methods include the use of commercially available vector network analyzers (VNA), that are usually bulky, or the custom fabrication of more compact instruments that are not oriented for the use at the point of care. As a result, the use of acoustic sensor platforms outside of specialized laboratories or in the field is not possible due to portability and cost limitations.

According to a preferred embodiment, an instrumentation scheme may be simplified significantly by introducing a method where a radio frequency (RF) generator and a detector communicate with a microcontroller and the recorded measurements are transformed to visualized graphs in real-time.

According to this preferred embodiment, the apparatus comprises of a RF generator, such as Phase-Locked Loop (PLL) or Direct Digital Synthesis (DDS), controlled by a microcontroller, configured to sweep from lower to higher frequencies in a short amount of time, while having the minimum amount of phase noise and jitter. Preferably, a band pass filter may be used for the rejection or attenuation of unwanted harmonics.

According to this preferred embodiment, the apparatus also comprises a RF detecting module, configured to detect gain and phase changes. Preferably, the detector is configured to reject any unwanted signals/noise and report the measurements to a microcontroller or a microprocessor for further analysis. Preferably, the detector is configured to work within the frequency range of the acoustic sensor and retain measurements linearity. Preferably, the detector complementary circuitry, such as attenuators and matching networks, exploit the maximum available dynamic range of the detector. For example, input impedance matching and attenuation on the ports of the detector may be matched with the characteristics of the acoustic sensor.

According to this preferred embodiment, the apparatus also comprises an ultra-low noise analog to digital converter (ADC) that is configured to transform analog readings from a RF detector and send them in a digital format to a microcontroller or a microprocessor. Preferably, the ADC module has more that 12-bit resolution, in order to report precise measurements.

According to this preferred embodiment, as it is presented in Figure 5, the apparatus also comprises a microcontroller or a microprocessor unit that may be configured to perform multiple tasks. Preferably, the microcontroller sends the recordings in real time in any format/medium, such as a screen (20), a memory card, or a computer/smartphone. Preferably, the microcontroller is configured to perform an automated program, to sweep throughout the working spectrum and selecting the best working frequency of the acoustic sensor. Preferably, the microcontroller is configured to communicate with all the aforementioned electronic apparatus, such as heating element, means configured to inject the sample, RF detector and RF generator, and run an automated program to perform the desired assay/analysis/test/experiment.

Therefore, another aspect of the present invention is a method for generating the signal that is required for the acoustic sensor to operate and monitoring real-time changes using a simplified instrumentation that can communicate with handheld and personal device, like smartphones and computers.

The advantage of the proposed apparatus and method, is the lack of sophisticated and complicated instruments that are used in prior art approaches. Therefore, the present invention, is a more user friendly, easy to fabricate and a low cost approach that can report to any available medium, such as a smartphone, the results of an assay precisely and in a short amount of time. The apparatus and methods of the present invention can be used as a portable or bench- top platform. Preferably, the platform is portable and has low-energy consumption for use in any environment and application.

The effectiveness of the present invention is the same as that of prior art approaches, in which a microfluidic cell is placed directly on the sensor surface. However, the present invention does not require any sort of precision engineering or microfabrication methods that increase significantly the final cost and the complexity of the apparatus.

Another advantage of the present invention is that, after each test that may be performed on the apparatus, the used flow strip and absorbent pad, if present, can be discarded without considering any special disposal handlings. Preferably, a single use flow strip may be used for each test. Furthermore, means for positioning the strip may be reusable, and could be made from a resistant material that can be decontaminated from the reagents and the under-investigation molecules/targets, using various means, for example, chemical treatment, radiation or biological treatment. Furthermore, the acoustic sensor may be reusable or it can be discarded after each experiment.

The present inventors have surprisingly found that when the flow strip is placed above the acoustic sensor, so that the flow strip and the sensing area of the acoustic sensor form parallel plates of a capillary space there is a negligible attenuation of the acoustic wave energy, since the flow strip does not come in contact with the acoustic sensor (Figure 6). This is evident by comparing the maximum amplitude values from Figure 6a (without flow strip) and Figure 6b (with flow strip), where without the strip the amplitude maximum is 1122mV (millivolts) (Figure 6a, pinpointed), while with the flow strip the amplitude maximum is 1117.5mV (Figure 6b, pinpointed). Thus, the difference is 4.5mV which equals to 0.15 dB (based on the fact that the RF detector, used in the measurement, has a measurement scaling of 30mV/dB). The flow strip does not come into contact with the sensing area of the sensor, thus the acoustic wave propagates freely along the sensing area, without meeting any absorbent materials, such as the walls of a microfluidic flow cell or any confinement structure. In the approaches of the prior art, upon the formation of the capillary interface, the signal is attenuated due to the mismatch of the dielectric constants of the liquid and the piezoelectric substrate. On the other hand, the total signal losses of the present invention are significantly less than in the prior art approaches, and therefore, any optimization for increasing the specificity or sensitivity of the assay, has to be performed solely on the acoustic sensor in use, and not on both the acoustic sensor and the microfluidic apparatus.

Examples

Example 1

Detection of DNA ladder in liquid sample

An example of biomolecule detection with an apparatus according to the present invention, is presented in Figure 7. Prior to each experiment, the apparatus was thoroughly cleaned with proper solutions, such as solvents (e.g., ethanol) and buffer solutions, and dried out with air or nitrogen, in order to remove any contaminants from the surface of the acoustic device and the surrounding area. Then, the apparatus performs a frequency sweep near the working frequency of the acoustic sensor without the flow strip placed on top of the sensor. The apparatus obtains the response of the sensor in a 3 MHz to 5MHz span and locks the oscillator frequency on the frequency that the amplitude is maximized. After finding the working frequency, the apparatus enters the real-time monitoring mode and the flow strip is placed on top of the acoustic device, to form a capillary space between the flow strip and the sensor. After that, buffer solution is injected with a constant flow to form the capillary interface. While monitoring the acoustic signal in real-time, the formation of the capillary effect results in the attenuation of the signal. The frequency sweep procedure (Figure 7a) is repeated again and a new working frequency is found. After that, the setup enters again the real-time monitoring mode and the test begins. An indicative real-time experiment with the adsorption of two different concentrations (1 pg/mL and 10 pg/mL) of DNA ladder (N3231S, New England Biolabs, UK) on a functionalized with PLL(20)-g[3.5]- PEG(2) (Poly- L-Lysine grafted with Polyethylene glycol side chains) (SuSoS, Switzerland) surface, is presented in Figure 7b. In this experimental procedure, a polyester-backed nitrocellulose flow strip was placed on top of a four array SAW quartz sensor with a working frequency of 153.2 MHz. Trizma hydrochloride solution (Tris) (T2319, Sigma-Aldrich, USA) with a concentration of 10mM was used as the buffer solution of the experiment. The flow rate was adjusted at 15pL/min, while the experimental temperature was constant at 25°C. Upon the formation of the capillary effect by injecting Tris buffer solution, PLL-g-PEG solution with a concentration of 0.1 mg/mL (diluted in Tris 10mM), was used to functionalize the acoustic device sensing area. PLL-g-PEG, is a positively charged polymer which prevents the nonspecific binding of proteins and other macromolecules, while it allows the adsorption of the negatively charged DNA molecules. As it is depicted in Figure 7b, upon the injection of PLL- g-PEG solution at 165 seconds, both amplitude and phase change due to the adsorption of PLL-g-PEG on the sensor surface. After the following buffer rinsing, the two different DNA ladder samples were injected sequentially with buffer rinsing in between (Figure 7b).

Example 2

Detection of Bovine Serum Albumin in a liquid sample

A second example of biomolecule detection with an apparatus according to the present invention, it is presented in Figure 8. Prior to each experiment, the apparatus was thoroughly cleaned with proper solutions, such as detergents and buffer solutions, and dried out with air or nitrogen, in order to remove any contaminants from the surface of the acoustic sensor and the surrounding area. Then, the apparatus performs a frequency sweep near the working frequency of the acoustic sensor without the flow strip placed on top of the sensor. The apparatus obtains the response of the sensor in a 3 MHz to 5MHz span and locks the oscillator frequency on the frequency that the amplitude is maximized. After finding the working frequency, the apparatus enters the real-time monitoring mode and the flow strip is placed on top of the acoustic device, to form a capillary space between the flow strip and the sensor. After that, buffer solution is injected with a constant flow to form the capillary interface. While monitoring the acoustic signal in real-time, the formation of the capillary effect results in the attenuation of the signal. The frequency sweep procedure (Figure 8b) is repeated and a new working frequency is found. After that, the apparatus enters again the real-time monitoring mode and the test begins. An indicative real-time experiment with the adsorption of different concentrations of Bovine Serum Albumin (BSA, Sigma-Aldrich, USA) (from 0.01 mg/mL up to 40 mg/mL), is presented in Figure 8b. In this experimental test, a polyester-backed nitrocellulose flow strip was placed on top of a four array SAW quartz sensor with a working frequency of 153.2 MHz. Phosphate-Buffered Saline (PBS, P4417, Sigma-Aldrich, USA) with a concentration of 10mM was used as the buffer solution of the experiment. The flow rate was adjusted at 15pL/min, while the experimental temperature was constant at 25°C. Upon the formation of the capillary effect by injecting PBS buffer solution, Poly-L-Lysine (PLL, P8920, Sigma-Aldrich, USA) solution with a concentration of 0.1 mg/mL (diluted in PBS 10mM), was used to functionalize the acoustic device sensing area. PLL, is a positively charged polymer which allows the non-specific adsorption of negatively charged biomolecules, such as BSA. As it is depicted in Figure 8b, upon the injection of PLL solution at 180 seconds, both amplitude and phase change due to the adsorption of PLL on the sensor surface. After the following buffer rinsing, different concentrations of the BSA protein were injected sequentially with buffer rinsing in between (Figure 8b).

References

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Claims

1. An apparatus for the detection of a biomolecule in a liquid sample, wherein the apparatus comprises an acoustic sensor (6), a removable flow strip (7), means (8, 9, 10, 12) configured to position the flow strip (7) above the acoustic sensor (6) so that the flow strip (7) and the sensing area of the acoustic sensor (6) form a top and a bottom parallel plate of a capillary space, wherein the capillary space does not comprise side plates and wherein the width of the flow strip is equal or smaller than the width of the sensing area of the acoustic sensor, means for injecting the liquid sample onto the flow strip, and a detector configured to detect the signal of the acoustic sensor.

2. The apparatus according to claim 1 , wherein the means for injecting the liquid sample are configured to inject a constant flow of the liquid sample.

3. The apparatus according to claim 1 or 2, wherein the apparatus further comprises an absorbent pad (14) which is attached to the flow strip (7) and is configured to collect the liquid from the flow strip (7) downstream of the acoustic sensor (6).

4. The apparatus according to any one of the preceding claims, wherein the sensing area of the acoustic sensor (6) is functionalized with a molecule that can bind specifically to the biomolecule of the liquid sample.

5. The apparatus according to any one of the preceding claims, wherein the apparatus further comprises a sample preparation station (1).

6. The apparatus according to any one of the preceding claims, wherein the apparatus further comprises a heat exchanger (2, 3) for increasing and/or reducing the temperature of the sample.

7. The apparatus according to any one of the preceding claims, wherein the acoustic sensor (6) is a bulk acoustic wave sensor, or a surface acoustic wave sensor.

8. The apparatus according to any one of the preceding claims, wherein the flow strip (7) is made from metal, glass, plastic, organic polymer, or inorganic polymer.

9. The apparatus according to any one of the preceding claims, wherein the flow strip (7) is made from nitrocellulose, preferably, from polymer-backed nitrocellulose.

10. The apparatus according to any one of the preceding claims, wherein the flow strip (7) is disposable.

11 . The apparatus according to any one of the preceding claims, wherein the apparatus further comprises a radio frequency generator and a microcontroller, which are configured to communicate with the detector.

12. The apparatus according to any one of the preceding claims, wherein the apparatus is portable.

13. A method for the detection of a biomolecule in a liquid sample, wherein the method comprises the steps of a) injecting the liquid sample onto a flow strip, wherein the flow strip is removably positioned above the sensing area of an acoustic sensor so that the flow strip and the sensing area of the acoustic sensor form a top and a bottom parallel plate of a capillary space, wherein the capillary space does not comprise side plates and wherein the width of the flow strip is equal or smaller than the width of the sensing area of the acoustic sensor, and b) detecting the signal of the acoustic sensor.

14. The method according to claim 13, wherein the flow rate of the liquid sample on the flow strip is from 2pL/min to 50 pL/min.

15. The method according to claim 13 or 14, wherein the gap between the flow strip and the sensing area of the acoustic sensor is from 0.1 mm to 0.5mm.