EP4609950A1

EP4609950A1 - Microfluidic device for detection of bioburden

Info

Publication number: EP4609950A1
Application number: EP24160416.4A
Authority: EP
Inventors: Sam George Vere CHARLTON; Eleonora SECCHI
Original assignee: Eidgenoessische Technische Hochschule Zurich ETHZ
Current assignee: Eidgenoessische Technische Hochschule Zurich ETHZ
Priority date: 2024-02-29
Filing date: 2024-02-29
Publication date: 2025-09-03
Also published as: WO2025181064A1

Abstract

A microfluidic device comprises a flow channel (10) defining a flow direction (F), an array (20) of pillars (23) arranged in the flow channel (10), the array (20) comprising a supporting row (21) of pillars, the pillars of the supporting row (21) being separated by supporting row gaps (24) having a supporting row gap width (g ₁), and a membrane (30) comprising colloidal particles which are packed against the supporting row. At least some of the colloidal particles are larger than the supporting row gap width. Also disclosed is a method of manufacturing the device and a method of detecting bioburden using the device.

Description

TECHNICAL FIELD

The present invention relates to a microfluidic device that is configured to detect bioburden, to a method of manufacture of a microfluidic device, and to a method of operating a microfluidic device.

PRIOR ART

Product quality and safety during biomanufacturing rely on monitoring for the absence of contaminating microbial agents known as bioburden. Bioburden detection is classified into off-line and on/in-line methods. While off-line methods still dominate the market, they are labor-intensive and time-consuming. For example, agar plating, which is the current industry standard method, has a time to result of 5-14 days. This causes a significant bottleneck in the production and release of biomanufactured products. The lack of on/in-line real-time bioburden sensors stands as a barrier to the adoption of continuous automated biomanufacturing and makes spatially locating contamination sources within a pipeline challenging, leading to the disposal of product and economic loss.
Therefore, novel on-line or in-line bioburden sensor technologies are paramount for unlocking production capacity, improving manufacturing efficiency and accelerating product release for patients and consumers.

SUMMARY OF THE INVENTION

In a first aspect, it is an object of the present invention to provide a device that enables real-time detection of bioburden in on/in-line applications. It is another object of the present invention to provide a device that enables the detection of bioburden at low cost and without complex and expensive equipment.
This object is achieved by a microfluidic device according to claim 1.
A microfluidic device is disclosed, comprising:

a flow channel defining a flow direction;
an array of pillars arranged in the flow channel, the array comprising a supporting row of pillars, the pillars of the supporting row being separated by supporting row gaps having a supporting row gap width; and
a membrane comprising colloidal particles which are packed against the supporting row upstream of the supporting row relative to the flow direction, all or part of the colloidal particles being larger than the supporting row gap width.

The proposed microfluidic device has simple configuration and can be readily manufactured with a high degree of automation and at low cost. Bioburden can be detected with high sensitivity and without complex and expensive equipment by monitoring the flow resistance of the membrane ("hydraulic sensing modality"). As will be described in more detail below, other sensing modalities such as electrical impedance detection ("electrical sensing modality") and/or florescence detection ("fluorescent sensing modality") can be added or may be employed instead. Being a microfluidic device, the device can be constructed in a sufficiently compact manner to enable installation at multiple points within a manufacturing pipeline, which offers the possibility of spatially localizing contamination. The properties of the membrane can be easily tailored to meet different requirements by appropriately choosing the materials and the size distribution of the colloidal particles. The colloidal particles can be functionalized to further tailor their properties, e.g., for selectively detecting specific target species. If the colloidal particles are functionalized with moieties that exhibit fluorescence in the presence of at least one target species, this can be used to implement the fluorescent sensing modality. Once bioburden has been detected, biomaterial can be readily isolated for further analysis by backflushing a fluid through the membrane in a direction opposite to the flow direction.
In order to provide stability to the membrane, the membrane is preferably sintered such that the colloidal particles in the membrane are at least partially fused. To facilitate sintering, the membrane preferably comprises at least 40% by number of colloidal particles that comprise a thermoplastic polymer, for example, polystyrene (PS).
In addition to the supporting row of pillars, the pillar array may further comprise a backflush row of pillars, the backflush row being arranged upstream of the supporting row at a predefined distance with respect to the flow direction. The backflush row provides added stability when a flow against the flow direction is established, e.g., during backflushing operations. The pillars of the backflush row are separated by backflush row gaps having a backflush row gap width that is larger than the supporting row gap width, preferably by a factor of at least 2, more preferably by a factor of at least 4. The membrane extends upstream of the supporting row preferably at least all the way to the backflush row, filling the entire region between the backflush row and the supporting row. In some embodiments, the membrane is exclusively arranged between the supporting row and the backflush row. In other embodiments, the membrane may project upstream beyond the backflush row.
In order to facilitate the assembly of the membrane, it is advantageous if at least 20% by number of the colloidal particles in the membrane have a size that is larger than the supporting row gap width, preferably by at least 5%.
In some embodiments, the membrane may comprise at least two species of colloidal particles, the species having different compositions and/or size distributions. For example, the membrane may comprise polymeric particles, e.g., polystyrene particles, in combination with inorganic particles, e.g., silica particles. As another example, the membrane may comprise particles having similar composition, but different sizes, e.g., at least two differently sized fractions of polystyrene particles. As yet another example, the membrane may comprise dielectric particles in combination with electrically conductive particles. The two or more species may be present throughout the membrane, or the membrane may have a layered structure, each layer comprising predominantly one of the species. Thereby, great flexibility is obtained in tailoring the properties of the membrane. One or more of the species may be functionalized.
For monitoring the flow resistance of the membrane (hydraulic sensing modality), a bioburden detection system may be provided, comprising the microfluidic device of the first aspect and a flow rate sensor to determine a flow rate through the membrane. In addition or in the alternative, the system may comprise a differential pressure sensor for determining a pressure difference across the membrane, e.g., a pressure difference between a region of the flow channel upstream of the membrane and a region of the flow channel downstream of the membrane. The system may further comprise electronic circuitry configured to determine a change of flow resistance across the membrane based on the flow rate and/or pressure difference to detect the bioburden.
In addition to or instead of monitoring flow resistance across the membrane, it is possible to detect bioburden by monitoring an electrical impedance in the microfluidic device (electrical sensing modality). To this end, the microfluidic device may comprise:

a working electrode proximate to the membrane;
a counter electrode proximate to the membrane; and
optionally, a reference electrode, e.g., an AgCl reference electrode.

The microfluidic device may further comprise contact pads for establishing contact between these electrodes and external electronic circuitry.
A corresponding bioburden detection system may further comprise electronic circuitry for carrying out electrical impedance measurements, in particular, electrochemical impedance spectroscopy (EIS), using the working electrode, counter electrode and, optionally, reference electrode to determine impedance changes in the microfluidic device due to particle deposition on the membrane.
In some embodiments, the membrane may comprise electrically conductive particles that cause the membrane or at least a portion of the membrane to be electrically conductive. This is particularly advantageous if electrical impedance measurements are to be carried out.
The membrane may optionally comprise particles that have been functionalized with an agent that facilitates specific binding of at least one target species to the functionalized particles.
For implementing the fluorescent sensing modality, the particles may have been functionalized with an agent that carries a fluorescent moiety whose fluorescence is quenched as long as a target species is not present and which exhibits fluorescence once the target species is present. For example, the fluorescent moiety may be cleaved from the agent as a result of binding of the target species to the agent and may exhibit fluorescence once it has been cleaved.
The colloidal particles and/or the flow channel walls may optionally be coated with an anti-adhesion or anti-fouling agent such as bovine serum albumin (BSA) or PLL-g-PEG (a random graft co-polymer with a poly(L-lysine) backbone and poly(ethylene glycol) side-chains).
The microfluidic device may have different channel configurations. In some embodiments, the microfluidic device comprises an inlet port for supplying a fluid to the flow channel and an outlet port for removing the fluid from the flow channel, wherein the membrane is arranged in the flow channel in such manner that the fluid that enters the flow channel through the inlet port traverses the membrane before it leaves the flow channel through the outlet port. This configuration will in the following be called the "normal" configuration. In other embodiments, the microfluidic device comprises a main inlet port for supplying a fluid to the flow channel during operation of the microfluidic device, a main outlet port for removing a first portion of the fluid from the flow channel during operation, the first portion having flowed past the membrane without traversing the membrane, and an auxiliary (or "tangential") outlet port downstream of the membrane for removing a second portion of the supplied fluid from the flow channel, the second portion having traversed the membrane. Optionally, the device may further comprise an auxiliary inlet port for supplying fluid to the flow channel during assembly of the membrane.
The microfluidic device may be configured as an exchangeable cartridge, enabling easy replacement in a bioburden detection system.
In a second aspect, a method of manufacturing a microfluidic device is provided, in particular, for manufacturing the microfluidic device of the first aspect. The method comprises:

providing a microfluidic chip comprising a flow channel defining a flow direction and an array of pillars arranged in the flow channel, the array comprising a supporting row of pillars, the pillars of the supporting row being separated by supporting row gaps having a supporting row gap width;
creating a flow of colloidal particles dispersed in a carrier fluid through the flow channel along the flow direction, at least some of the colloidal particles being larger than the supporting row gap width, to cause flow-directed assembly of a membrane that comprises the colloidal particles; and
sintering the membrane to at least partially fuse the colloidal particles in the membrane.

The method may comprise applying a flow rate ramp to increase a packing density of the colloidal particles in the membrane during or after assembly of the membrane, but before sintering. The method may comprise functionalizing at least some of the colloidal particles.
Functionalization may be carried out before or after assembly of the membrane.
In a third aspect, a method of detecting bioburden using the microfluidic device of the first aspect is disclosed. The method comprises:

causing a flow of a fluid through the microfluidic device; and
monitoring a flow resistance through the membrane (first sensing modality) and/or an electrical impedance in the microfluidic device (second sensing modality) and/or fluorescence at the membrane (third sensing modality) to detect the bioburden.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,

Fig. 1: shows a schematic top view of an embodiment of a portion of a microfluidic device;
Fig. 2: shows a schematic side view of the embodiment in Fig. 1;
Fig. 3: shows a schematic front view of the embodiment in Fig. 1;
Fig. 4: shows a normal configuration of a microfluidic device;
Fig. 5: shows a tangential configuration;
Fig. 6: shows a variant of the tangential configuration;
Fig. 7: shows functional sketches illustrating the use of the tangential configuration;
Fig. 8: shows functional sketches illustrating the use of the variant of the tangential configuration;
Fig. 9: shows a diagram in which normalized flow rate through the membrane is plotted as a function of pressure;
Fig. 10: shows a diagram in which normalized flow resistance of the membrane is plotted as a function of the membrane composition;
Fig. 11: illustrates a first sensing modality for detecting bioburden with a microfluidic device having a normal configuration;
Fig. 12: illustrates the first sensing modality for detecting bioburden with a microfluidic device having a tangential configuration;
Fig. 13: shows microscopic images of a portion of a microfluidic device which show bacterial growth on the membrane as a function of time;
Fig. 14: shows a diagram in which normalized flow resistance of the membrane is plotted as a function of the number of particles captured on the membrane (left) and a detection limit is derived from this plot (right);
Fig. 15: shows a schematic top view of an embodiment of a microfluidic device configured for EIS, thus implementing a second sensing modality;
Fig. 16: shows a schematic side view of the embodiment of Fig. 15;
Fig. 17: shows a first electrode design;
Fig. 18: shows a second electrode design;
Fig. 19: shows a flow diagram illustrating a method of manufacture; and
Fig. 20: shows a flow diagram illustrating a method of detecting bioburden.

DESCRIPTION OF PREFERRED EMBODIMENTS

Definitions

The term "microfluidic device" is to be understood as referring to a device that comprises at least one flow channel having at least one lateral dimension (in particular, the channel height perpendicular to a device plane of the microfluidic device) that is not more than 1 millimeter.
The term "colloidal particle" is to be understood as referring to a solid particle that can be suspended in a carrier fluid to form a colloidal suspension (a sol in case of a carrier liquid or a solid aerosol in case of a carrier gas). Preferably, the colloidal particles used in the present context have a size between about 200 nm and about 100 µm, more preferably between about 1 µm and about 20 µm.
The term "flow channel" is to be understood as referring to a hollow structure that enables a flow to be established along a flow direction. While a flow channel may have arbitrary cross-sectional shape, it is advantageous in terms of fabrication if the flow channel has essentially rectangular cross-sectional shape. Preferably, the flow channel is a microfluidic flow channel, i.e., at least one lateral dimension of the flow channel, in particular, its height, preferably is not more than 1 millimeter.
The term "membrane" is to be understood as referring to a porous structure that extends across the entire cross section of the flow channel and allows a carrier fluid to pass while retaining solid particles beyond a certain size.
The term "pillar" (or "column") is to be understood as referring to an elongated structure that is arranged in the flow channel and extends transverse to the flow direction. Preferably, each pillar is vertical, i.e., it extends in a height direction perpendicular to a device plane of the microfluidic device. Preferably, each vertical pillar spans the entire height of the flow channel from the bottom limiting wall to the top limiting wall of the flow channel. A pillar preferably has cylindrical shape. However, other shapes are possible as well, for example the shape of a straight polygonal prism.
The term "row of pillars" is to be understood as referring to an essential linear arrangement of pillars that are similarly spaced. A row may be straight or curved. The term "row" also includes arrangements in which the pillars are staggered in a zigzag fashion. A row of vertical pillars preferably extends essentially across the entire width of the flow channel, allowing a flow through the row of pillars along the flow direction only through gaps that have a gap width corresponding to the distance between neighboring pillars.
A direction "transverse to the flow direction" is to be understood as being a direction that subtends an angle of at least 60° to the flow direction. Preferably, a transverse direction is essentially perpendicular to the flow direction.

Determination of particle and pore sizes

The size of colloidal particles that have not yet been integrated into the membrane may be determined by scanning electron microscopy (SEM). The following protocol may be used: First, a dilute 0.001% wt particle suspension is sonicated for 15 mins to break down particle aggregates, yielding a homogeneously dispersed suspension. Then, 10 µL of the particle suspension is deposited onto a UV treated SiO₂ wafer and left for 2 hours to evaporate, resulting in a dispersed monolayer of particles. The dried wafer is then sputter coated with 0.5 nm Pt. Imaging is performed using a scanning electron microscope. The particle size distribution is calculated from the images using a circle Hough Transform.
For determining the size of particles that have been integrated into the membrane and of the pores in the membrane, confocal scanning microscopy may be used. The following protocol may be employed: The device comprising the membrane is transferred to an inverted confocal scanning laser microscope. If the particles are functionalized with a fluorophore, the excitation and emission settings are set for the fluorophore. The pinhole size is set to 1 Airy unit. A volume scan of the membrane is performed across the width of channel with a z step size of e.g. 0.5 µm. The images are then analyzed using standard image processing methods to obtain particle centroids and particle diameters. For non-fluorescent particles, a fluorescent dye (e.g., FITC) is flown through the membrane. The dye is then imaged using a volume scan across the width of the membrane. The images are analyzed using standard image processing methods to obtain the particle and pore size distributions.

General setup

An embodiment of a microfluidic device is illustrated in Figs. 1 to 3, which show a central portion of the device containing the membrane. The illustrated microfluidic device comprises a PDMS substrate 1 and a glass cover 2. An elongated microfluidic flow channel 10 having rectangular cross section with width w and height h is formed in the substrate 1 and covered at its top end by the cover 2. A fluid flow 11 is able to flow through the flow channel 10 along a flow direction F.
It is to be understood that the drawings in Figs. 1 to 3 are schematic and not true to scale. In particular, in practical applications, the ratio between the width w and the height h may be considerably larger than illustrated.
An array 20 of cylindrical pillars 23 is arranged in the flow channel 10. The pillars 23 are integrally formed by the PDMS substrate. Each of the pillars 23 extends vertically along a height direction, perpendicular to a device plane of the microfluidic device, the device plane corresponding to the drawing plane of Fig. 1. Each of the pillars 23 spans the entire height h of the flow channel 10 from the bottom of the flow channel all the way to the cover 2. The array 20 comprises two rows of pillars: a supporting row 21 and a backflush row 22. Each row extends across the entire width w of the flow channel 10. In the present example, each of the rows 21, 22 is straight, the pillars of each row being arranged along a straight line that is perpendicular to the flow direction F. In the present example, all pillars have equal shape and dimensions. However, the shapes and dimensions may be different between the two rows and even within each row.
The pillars of the supporting row 21 are separated by supporting row gaps 24 having a supporting row gap width g ₁. The pillars of the backflush row 22 are separated by backflush row gaps 25 having a backflush row gap width g ₂, which is larger than the supporting row gap width g ₁. The supporting row 21 and the backflush row 22 are separated by a distance d along the flow direction F.
A membrane 30 is arranged in the region between the supporting row 21 and the backflush row 22. The membrane 30 comprises colloidal particles, which are packed against the supporting row 21. For an illustration of colloidal particles packed against the supporting row 21, see Figs. 15 and 16, which will be discussed in more detail below. At least some of the colloidal particles are larger than the supporting row gap width g ₁, whereby the colloidal particles are prevented from escaping through the supporting row gaps 24. The backflush row 22 supports the membrane 30 during back flushing events.
It is to be understood that the number of pillars in each row may be considerably larger than illustrated.

Membrane assembly

The membrane 30 is created by causing a flow of a suspension comprising the colloidal particles along the flow direction F and allowing the membrane to assemble against the supporting row 21. This method will in the following be referred to as "flow-directed assembly". The colloidal particles in the suspension do not need to have uniform size and/or composition. In particular, the membrane may comprise colloidal particles that are smaller than the supporting row gap size g ₁ because larger particles that have already been retained by the supporting row 21 will prevent smaller particles from passing. The membrane may thus comprise colloidal particles that are smaller than the supporting row gap size g ₁ as long as at least some of the colloidal particles in the suspension have sufficiently large size to be retained by the supporting row 21. Preferably, at least 20% of the colloidal particles (by number) have a size that is larger than the supporting row gap width g ₁, said size preferably being at least 5% larger than the supporting row gap width.
Preferably all colloidal particles in the membrane 30 are smaller than the backflush row gap width g ₂ to ensure that the particles are able to pass through the backflush row gaps 25. However, it is also conceivable that particles larger than the backflush row gap width are added to the suspension once a sufficient number of colloidal particles have been retained by the supporting row 21 to completely fill the region between the supporting row 21 and the backflush row 22.
During or after membrane assembly, the flow rate can be varied to modulate the packing of the colloidal particles in the membrane. For example, a flow ramp with a continuously increasing flow rate can be used to compact the packing. The procedure produces a flat packing structure, as flow is redirected away from regions with higher thickness due to the increased flow resistance of such regions.
After membrane assembly, the membrane 30 is sintered by heating the membrane to a temperature that is sufficiently high to cause the colloidal particles in the membrane to at least partially fuse, conferring mechanical stability to the membrane. To facilitate sintering, it is advantageous if the membrane comprises at least 40% by number of colloidal particles made of a thermoplastic polymer. The temperature during sintering should be near the glass transition temperature of the polymer of the colloidal particles and below its melting temperature. In particular, polystyrene particles may be used. On the other hand, the temperature during sintering should preferably be below the glass transition temperature of the material of the microfluidic chip to avoid that the flow channels deform during sintering.

Flow configurations

Figs. 4-6 illustrate three exemplary configurations of the microfluidic device. For simplicity, the pillar array is not shown in these figures.
In the configuration of Fig. 4, the microfluidic device has a single inlet port 12 and a single outlet port 13, without other inlet or outlet ports. The flow channel 10 extends from the inlet port 12 to the outlet port 13. The entire flow that enters the flow channel 10 through the inlet port 12 passes through the membrane 30 before the flow leaves the flow channel at the outlet port 13. This configuration will in the following be called the "normal flow configuration".
In the configuration of Fig. 5, the microfluidic device has two inlet ports, which may be called a "main" inlet port 14 and an "auxiliary" inlet port" 16, as well as two outlet ports, which may be called a "main" outlet port 15 and an "auxiliary" or "tangential" outlet port 17. A first flow channel, which will in the following also be called the "main channel", extends from the main inlet port 14 to the main outlet port 15, and a second flow channel extends from the auxiliary inlet port 16 to the auxiliary outlet port 17. The channels intersect to form a cross. The membrane 30 is arranged in the second flow channel immediately downstream of the intersection of the two channels. This configuration will in the following be called the "tangential flow configuration", and the section of the second flow channel that extends between the intersection and the tangential outlet port 17, containing the membrane 30, will in the following be called the "tangential outlet channel".
A simplified variant of the tangential flow configuration is illustrated in Fig. 6. Here, the auxiliary inlet port has been left away.
Operation of the tangential flow configuration is now explained with reference to Fig. 7. During assembly of the membrane, the main inlet port 14 and the main outlet port 15 are closed. A flow of a colloidal suspension is created from the auxiliary inlet port 16 to the tangential outlet port 17 to cause flow-directed assembly of the membrane 30. During normal operation, a fluid flow is caused from the main inlet port 14 to the main outlet port 15. If a positive pressure difference exists between the main inlet port 14 and the tangential outlet port 17, a portion of the flow will pass through the membrane 30 and will leave the device at the tangential outlet port 17.
As illustrated in Fig. 8, operation of the simplified variant with only three ports is similar. During assembly of the membrane, the main outlet port 15 is closed, and flow of a colloidal suspension is caused from the main inlet port 14 to the auxiliary outlet port 17 to cause flow-directed assembly of the membrane 30. Normal operation after creation of the membrane 30 is identical to the configuration of Fig. 7.

Considerations concerning membrane assembly

As the construction of the membrane relies upon the pillar layout and the height and width of the channel, the membrane surface area can be tuned by selecting the height and/or width of the channel. The limiting step here is the aspect ratio of the pillars, which may be limited by the available microfabrication techniques. Preferably, the pillar array is arranged at a certain a distance from the inlet port that is used for membrane assembly to ensure a well-controlled velocity profile of the flow of the suspension containing the colloidal particles. The channel may be pre-treated with a solution of BSA or any other suitable anti-adhesion agent to reduce unintended adhesion of colloidal particles to the channel surface.
Assuming that the membrane is composed of particles having uniform radius r ₁, the number N ₁ of particles in the membrane can be calculated from the desired membrane thickness t (which may be identical with the distance d, but may also be greater than that distance if the membrane is to extend beyond the backflush row 22), the width w and the height h of the channel and the random packing limit of spheres, ϕ_rpl ≈ 0.68, as follows: $v = twh,$ $N_{1} = \frac{3 v}{ϕ_{rpl} \cdot (4 {πr}_{1}^{3})} .$
The number N ₁ can be calculated beforehand, and the corresponding number of colloidal particles can be injected into the flow channel to assemble the membrane. Instead of calculating this number beforehand, the thickness of the membrane can also be readily controlled by visually monitoring the thickness of the membrane during flow-directed assembly and slowly adding more particles to the flow until the desired thickness has been reached.
For membranes composed of two species of particles having different radii r ₁ and r ₂, the following equation can be used to calculate the numbers N ₁ and N ₂ of the particles, assuming the ratio N ₁/N ₂ is known: $v = ϕ_{rpl} \cdot (\frac{4 {πr}_{1}^{2}}{3} N_{1}) + ϕ_{rpl} \cdot (\frac{4 {πr}_{2}^{2}}{3} N_{2}) .$
This can be readily generalized to membranes comprising particles having more than two different radii.
The proposed process enables the fabrication of stable membranes embedded directly within microfluidic geometries (e.g., from 10 µm to 1000 µm) with tuneable surface area and pore size distributions (e.g., 50 nm to 25 µm) and allows the incorporation of multiple materials with distinct properties and functionalities into the membrane. The membranes inherently offer a substrate for bacterial growth and subsequent biofilm formation. The bioburden can be cultured in situ.

Example

Microfluidic chips were fabricated with both the normal configuration (Fig. 4) and with the simplified variant of the tangential configuration (Fig. 6). In both configurations, the flow channels had a height of 25 µm. The flow channel of the normal configuration had a uniform width of 500 µm and a total length of 1.5 cm. The flow channels in the tangential configuration had a width of 250 µm. The channel portions connecting the inlet and outlet ports to the intersection were each 1 cm long.
The supporting row 21 and the backflush row 22 were formed by cylindrical pillars 23 having a diameter of 5 µm. The gap width between the pillars of the supporting row 21 was 4 µm, which corresponds to 80% the size of the largest colloid used for the membrane packing. The backflush row 22 had a gap width of 40 µm, which corresponds to 800% the size of the largest colloid used in the membrane packing.
In the normal configuration, the membrane had a thickness that corresponded to the distance d between the supporting row 21 and the backflush row 22.
In the tangential configuration, the backflush row 22 was placed either 10 µm or 50 µm downstream of the intersection between the main channel and the tangential outlet channel, and the membrane extended all the way from the intersection to the supporting row (for an illustration, see Fig. 12). As a result, the membrane had a thickness corresponding approximately to the distance d between the supporting row 21 and the backflush row 22 plus the distance between the intersection and the backflush row 22.
The geometries for each of the channels were fabricated using standard soft lithography methods on an SU8 wafer. Microfluidic chips were fabricated by mixing polydimethylsiloxane (PDMS) with a crosslinker at a ratio of 5:1 to prepare a PDMS mixture. This mixture was vigorously mixed and degassed for 1 hour. The PDMS mixture was then poured onto the SU8 wafer, degassed for 30 mins and transferred to an oven to cure at 80°C for 24 hours. Thereafter, the chips were cut, removed from the SU8 wafer and placed geometry side down on a hot plate at 120°C for 45 minutes. Then the inlet and outlet ports were created using a 1.5 mm surgical punch biopsy tool. The chips were cleaned with detergent and isopropanol, and each chip was bonded to a glass microscope slide that had been cleaned with isopropanol and blown with compressed air, the microscope slide forming a cover for the microfluidic channels in the chip. The chip and microscope slide were placed in a plasma oven for 1 minute before bonding and left to bake for 12 hours on a hot plate at 80°C.
Bare polystyrene (PS) colloidal particles (microParticles, Germany) with diameters of 5 µm and 3 µm and silica (SiO₂) particles with a diameter of 3 µm were used for membrane construction and underwent a two-step functionalization process. The first step was performed ex situ in suspension. Colloidal particles were diluted in 0.2 µm filtered distilled water to obtain a final concentration by weight of 0.0025%. The following number ratios between the 5 µm and 3 µm polystyrene particles were used: 1:1, 3:1 and 5 µm only. In some experiments, 5 µm polystyrene particles were mixed with 3 µm SiO₂ particles at a ratio of either 1:1 or 3:1.
The suspensions were vortexed for 10 seconds in screw top centrifuge tubes. Colloidal particles were washed in TRIZA buffer (Merck, Germany) twice and then resuspended in 2 mg/ml dopamine (Merck, Germany) in TRIZA buffer. The container was placed in a heating block set at 50°C for 18 hrs. Thereafter, the colloidal particles were washed in 0.2 µm filtered distilled water, vortexed to re-suspend and then sonicated for 1 hour. The second functionalization step was performed in-situ, after membrane construction, and is described below.
The bonded chips were degassed for 30 mins prior to membrane construction. Tygon tubing was used to connect the syringes to the microfluidic device (inner diameter 508 µm, outer diameter 1.524 mm, #AAD04103, Saint-Gobain). The following describes the assembly process of the membrane for the normally orientated microfluidic geometry with a membrane thickness of 50 µm. First, 0.5 ml of 0.2 µm filtered distilled water was backflushed through each channel at a rate of 200 µl/min from the outlet port using a 1 ml syringe (U-100, LUER, Codan), until all air bubbles were removed from the channel. After purging, the syringe was disconnected from the outlet tubing, while the tubing remained inserted within the outlet port. Then, for the inlet, a 1 ml syringe was filled with 0.2 µm filtered distilled water or 70% ethanol, the syringe was connected to the inlet tubing and purged, until a droplet formed on the outlet of the tubing. Then, the tubing was placed within a vial of the colloidal particles and the syringe was backflushed at a rate of 200 µl/min for a total volume of 20 µl. The inlet tubing was then inserted into the inlet port. After visual inspection to ensure an absence of air bubbles the inlet syringe was driven at a rate of 15 µl/min. The membrane was made by flowing colloidal particles until the membrane structure extended up to the backflush row of pillars, the total thickness of the membrane corresponding to the gap d between the two pillar rows. At this point, the chip was transferred to a preheated hot plate set to 90°C with the inlet and outlet tubing still inserted and the flow remaining on. The chip remained on the hot plate for 60 seconds to stabilize before the flow was stopped. The chip was left on the hot plate at 90°C for 20 mins. At this point the inlet and outlet tubing were removed, and the chip was left for a further 30 mins at 90°C. The inlet and outlet ports were then sealed, and the chip could be stored at room temperature.
The methodology to form the membrane in the simplified variant of the tangential configuration was broadly similar to the normal configuration, with few procedural modifications. First the chip was purged from the tangential outlet port, then the main outlet port was sealed. Afterwards the colloidal suspension was driven from the main inlet port using the same flow rates and volumes as for the normal configuration. The membrane thickness was equal to the distance between the pillar rows plus the distance between the backflush row and the intersection of the main channel with the tangential outlet channel. After membrane formation, the main outlet port was opened, and the flow rate was increased to 30 µl/min to remove particles which protruded from the interface to leave a flush interface with the main channel. Then, the chip was transferred to the hot plate as per the procedure for the normal configuration.
A second functionalisation step was performed on the formed membrane in-situ. The chip with the formed membrane was degassed and refilled using a flow rate of 15 µl/min and 50 µl of an aqueous suspension of 0.2 mg/ml PLL-g-PEG (Susos, Switzerland) in TRIZMA buffer (pH 8.5) until the chip was filled. The PLL-g-PEG had respective sizes of PLL 20 kDa grafted to PEG 5 kDa using a 3 kDa linker. The inlet and outlet ports of the chip were then sealed. The chip was then placed on a hot plate at 50°C for 20 hours to allow the PLL-g-PEG to react with the dopamine functionalised colloids. During this time the inlet and outlet ports of the chip were sealed, to prevent evaporation. After 20 hours the chip was rinsed with 0.2 µm filtered distilled water. The chips were then filled with sterile 10 mM HEPES buffer (Merck, Germany), the inlets and outlets were sealed, and the device was stored at 4°C.
The chip was then transferred to a microscope. Flow for each of the channels was driven using pressure controllers (Flow EZ, Fluigent) in parallel with a controller (LineUp, Fluigent). Pressure remained at a constant value (>150 mbar) throughout measurement. The sample was kept in a fluid reservoir upstream of the membrane channel and connected to the membrane channel using tubing. The flow rate at the outlet was measured with a flow rate sensor (Flow unit, Fluigent). Flow rate data were obtained at a frequency of 1 Hz using control software (Oxygen, Fluigent). At the end of each experiment, the flow rate data was analysed online using a custom analysis algorithm. Bioburden in the sample was detected from the increase in system hydraulic resistance, which was calibrated against known cell concentrations, which had been counted using a flow cytometer (CytoFLEX, Beckman Coulter).

Stability of the membrane

Fig. 9 shows a plot of the flow rate u through the membrane, normalized to the maximum measured flow rate u_max, as a function of the pressure difference P across the membrane for a forward flow along the flow direction F in the normal configuration. Values are plotted for both a 3:1 mixture (circles) and a 1:1 mixture (squares) of 5 µm and 3 µm sized polystyrene particles. For both mixtures, the plot shows a nearly linear dependence of flow rate as a function of pressure, demonstrating that the membrane remains fully intact at pressure differences up to at least 200 mbar.

Tunability of the membrane

Fig. 10 shows a plot of the flow resistance H_r of the membrane, normalized to a reference membrane, as a function of the composition and size distribution of the colloidal particles. The horizontal axis show the mixing ratio of 5 µm and 3 µm particles in percent by number. Flow resistance is plotted for 100% 5 µm polystyrene (PS) particles (open square at mixing ratio 100%), mixtures of 5 µm and 3 µm polystyrene particles at mixing ratios of 3:1 (open circle at mixing ratio 75%) and 1:1 (open circle at mixing ratio 50%), and mixtures of 5 µm polystyrene particles with 3 µm silica (SiO₂) particles at mixing ratios of 3:1 (open square at mixing ratio 75%) and 1:1 (open square at mixing ratio 50%). If mixtures of differently sized polystyrene particles were used, the flow resistance strongly depended on the mixing ratio. The dependence was much weaker for mixtures of polystyrene particles with silica particles.
These results demonstrate that the hydraulic properties of the membrane can be readily tuned by the choice of the composition and size distribution of the colloidal particles in the membrane. Further factors affecting the hydraulic properties are membrane thickness and surface functionalization.

Hydraulic sensing modality

Bioburden can be detected by monitoring the flow resistance across the membrane. This will in the following also be referred to as a "hydraulic sensing modality". To this end, the flow rate through the membrane may for example be measured at constant pressure, or the pressure difference across the membrane may be measured at constant flow rate.
The hydraulic sensing modality is illustrated in Fig. 11 (normal configuration) and Fig. 12 (tangential configuration). Bioburden 40 builds up on the membrane 30 and affects the flow resistance across the membrane.
In the normal configuration, the flow through the membrane may be measured by a flow rate sensor 41 that is arranged either downstream of the outlet port 13 (as pictured) or upstream of the inlet port 12. In the tangential configuration, the flow rate is advantageously measured downstream of the tangential outlet port 17. In both configurations, it is also conceivable to integrate a flow rate sensor directly into the microfluidic chip. In addition, the pressure difference across the membrane may be determined using an optional pressure sensor 42, which may be configured as a differential pressure sensor. The flow rate and/or the pressure difference may be fed to electronic circuitry 43, which determines changes of the flow resistance of the membrane 30 based on this information to detect the formation of bioburden 40 on the membrane.
Fig. 13 shows actual examples of bioburden buildup. The membrane was seeded with a dilute suspension (500 cells) of overnight culture of S. epidermidis 1457 modified with a gfp plasmid. After seeding, nutrient media, TBS, was driven at a constant pressure of 150 mbar through the membrane, and the flow rate at the outlet was measured using a microfluidic flow rate sensor. The chip was incubated at 37 °C, and imaging was performed using an epi-fluorescent microscope.
Fig. 14 illustrates the sensitivity of the hydraulic sensing modality. On the left of this figure, a plot of normalized flow rate u/u_max as a function of the number of 1 µm sized particles on a membrane having a surface area of 0.0125 mm² is shown for membranes consisting either of a 3:1 mixture of 5 µm and 3 µm polystyrene particles (squares) or of a 3:1 mixture of 5 µm polystyrene particles with 3 µm silica particles (circles). The limit of detection was estimated by determining the number of particles needed for a 1% reduction of the flow rate. In bother cases, the estimated detection limit was between 15 and 20 particles.
The sensitivity is tuneable by modulating the particle size distribution, pore size distribution, membrane composition and the membrane surface area. To contend with complex samples found in downstream product flows, non-specific protein and antibody binding may be suppressed using blocking buffer approaches. The device may be operated continuously in a closed system configuration, compatible with on-line detection. The membrane's mechanical integrity enables high-pressure backflow for the ejection of cells captured upon its surface for downstream compositional analysis .

Impedance sensing modality

A second possibility for bioburden detection is illustrated in Figs. 15 and 16. In these embodiments, the microfluidic device comprises a working electrode WE, a counter electrode CE, and, optionally, a reference electrode RE. The working electrode WE and the counter electrode CE may be patterned onto the glass cover 2. The reference electrode RE may be formed by a lateral channel in the substrate 1, filled with AgCl paste, which may be contacted via a lead on the glass cover 2.
With such an arrangement, electrical impedance measurements may be carried out, in particular, electrochemical impedance spectroscopy (EIS). EIS is a powerful technique for the analysis of interfacial properties related to bio-recognition events occurring at the electrode surface, such as antibody-antigen recognition, substrate-enzyme interaction, or whole cell capturing. The technique is summarized, e.g., in Magar, H.S., "Electrochemical Impedance Spectroscopy (EIS): Principles, Construction, and Biosensing Applications", Sensors 2021, 21, 6578, https://doi.org/10.3390/s21196578.
For carrying out impedance measurements, EIS control hardware 50 may be connected to the microfluidic device, e.g., via contact pads 51 formed on the cover 2. A small-amplitude ac stimulus (voltage or current), usually superimposed on a dc signal (voltage or current), may be applied to the electrodes and the resulting response (current or voltage, respectively) may be measured over a wide range of frequencies. Suitable EIS control hardware is well known in the field and is available commercially.
Two possible electrode geometries will be described in the following by the way of example.
The first electrode geometry is shown in Fig. 17. In this geometry, the working electrode WE and the counter electrode CE are interdigitated, extending across the channel width. The geometry of the electrode pads may be, e.g., between 10 - 50 µm in width with a typical separation of 10 - 20 µm. The exposed microelectrodes within this array overlap with the membrane interface, with a portion of the microelectrodes extending a distance of e.g. 5 - 20 µm upstream of the membrane interface. This electrode geometry may in particular be employed when the membrane is not electrically conductive.
The second electrode geometry, illustrated in Fig. 18, features a counter electrode CE (width 10 - 50 µm) upstream (10 - 20 µm) of the membrane, which crosses the channel width. The working electrode WE is 10 - 20 µm downstream of the counter electrode CE, directly below the membrane interface. This arrangement of electrodes is in particular useful if the membrane is electrically conductive, being in direct contact with the working electrode WE, or has a conductive layer in direct contact with the working electrode WE, making the membrane a porous conductive component.
Several possibilities exist for creating a membrane that is electrically conductive or comprises at least one electrically conductive layer. For example, metal-doped (e.g., Au- or Ag-doped) polystyrene particles may be included in the membrane packing. As another example, bare polystyrene particles may be used, which are then modified after membrane assembly using an electroless coating method to coat the particles with a metal like Au or Ag.
The metal-doped or metal-coated particles may further be functionalized, e.g., with aptamers, monoclonal antibodies and/or peptides, to enable species-specific detection of one or more selected target species via EIS.

Electrode fabrication

Planar gold electrodes may be fabricated by means of metal sputtering on glass wafers, which may then be used to cover the microfluidic chip comprising the flow channel. Contact pads and connecting leads for connecting the electrodes with the contact pads may be provided for connection to the external EIS control hardware. The connecting leads may be insulated within a passivation layer.
The planar gold electrodes may be fabricated using well-established techniques. In one example, an AZ positive photoresist is spun on a clean glass wafer, patterned by means of photolithography and developed with tetramethyl-ammonium hydroxide to obtain a patterned protective layer, complementary to the aforementioned electrode configurations. A 10-40 nm layer of gold/chrome is then sputtered on the wafer and, subsequently, the photoresist is lifted off using acetone leaving the patterned planar gold electrodes. Optionally, a passivation layer (of e.g. SiO₂) can be similarly selectively patterned through vapor deposition on top of the gold layer in order to insulate all the gold leads except for the sensing area and the contact pads for connection. After fabrication, the glass wafer is diced into individual glass slides.
These glass slides are used to close the PDMS chips in which the microfluidic channels have been formed. The slides may be aligned with the PDMS pillar chip using a stereoscope and assembled; bonding may be performed using plasma activation.
To compensate for changes in media conductivity, a reference electrode can be incorporated, e.g., upstream of the pillar array and the working and counter electrodes. The reference electrode may be fabricated by filling a channel that is upstream of the membrane with AgCl paste. The reference electrode channel may have a series of pillars at the interface with the main channel, to minimize the inclusion of AgCl paste within the main flow channel. The entrance to the reference electrode channel may then be connected to the EIS control hardware.

Biochemical functionalization of the membrane

The membrane can be fabricated with chemically well-defined particles, enabling facile functionalization, either pre-assembly (in the case of biochemical molecules which do not denature during sintering), or in-situ after flow assembly and sintering. For example, in the case of a membrane comprising particles having a gold shell, the gold shell particles can be functionalized in-situ using aptamers with a thiol group conjugated to the 5' side to target lipopolysaccharides. This may be performed by flowing an aptamer suspension through the chip after membrane fabrication. Furthermore, in-situ membrane functionalization with PLL-g-PEG brushes with biotinylated PEG side chains, for example, can be applied to add recognition sites to the PEG brushes for specific targeting of biological species, e.g., viruses, mycoplasma and bacteria.

Real-time sensing

As described above, the device may integrate at least one out of three sensing modalities; the first, hydraulic resistance, whereby captured bioburden is detected through a drop in membrane permeability, which is measured using a microfluidic flow sensor and/or pressure sensor. The second applies electro impedance sensing directly to the membrane, which may be detected using EIS control hardware like a micro-oscilloscope module. The third sensing modality detects fluorescence upon binding of a target species to the membrane, which has been appropriately functionalized.
The construction methodology employed allows for the seamless integration of all three sensing modalities. Together this sensing platform enables on-line detection of bioburden (bacteria and fungi) in real-time. Furthermore, the on-line platform enables installation of bioburden sensing at multiple locations along a biomanufacturing pipeline, e.g., upstream for influent media, and downstream after centrifugation and initial purification steps. This enables spatial location of potential contaminant sources, providing the facility operators with the information required to rescue a product batch, which is not feasible with prior-art methods that operate off-line.
The platform safeguards against false positive/false negative results by enabling up to three parallel sensing modalities on a single surface, whilst simultaneously capturing bioburden, which can be back-flushed and isolated for downstream sequencing analysis. The platform can be operated without the addition of reagents or fluorescent probes, simplifying the operation of the device, and removing the need for skilled technicians to operate the device. If only the hydraulic and electrical sensing modalities are employed, no complex optical components are required.

Flow charts

Fig. 19 shows a flowchart that summarizes an example of a method of manufacturing a microfluidic device in accordance with the present disclosure. In step 61, a microfluidic chip comprising a flow channel with a pillar array is fabricated. In step 62, electrodes are applied to a glass cover, and the flow channel is closed using the glass cover such that the electrodes face the flow channel at the desired positions. In step 63, a suspension of colloidal particles is injected into the flow channel to cause flow-directed assembly of a membrane. In step 64, an optional flow rate ramp is carried out to increase the packing density of the membrane. In step 65, the membrane is sintered. In step 66, the sintered membrane is subjected to post-processing and/or functionalization operations.
Fig. 20 shows a flowchart that summarizes an example of a method of operating a bioburden detection system in accordance with the present disclosure. In step 71, the bioburden detection system is installed in the fluidic system to be monitored. In step 72, a replaceable cartridge comprising the microfluidic device with the membrane is inserted into the bioburden detection system. In step 73, a continuous flow of a fluid is caused through the microfluidic device. In step 74, flow resistance and/or electrical impedance and/or fluorescence of the membrane are monitored to detect bioburden on the membrane. If bioburden is detected in decision step 75, a reverse flow of a carrier liquid through the membrane is created to backflush and isolate the bioburden for further analysis. Otherwise, operation is continued in step 77. Once the cartridge has reached its end of life, it replaced in step 78.

Modifications

Various modifications to the above examples are possible. For example, instead of producing a microfluidic chip using soft lithography of a material like PDMS, the chip may be produced by injection molding of a thermoplastic material. In this case, the glass transition temperature of the chip material should be higher than the glass transition temperature of the thermoplastic colloidal particles to avoid channel deformation during the sintering step. Instead of using bare particles for membrane construction, pre-functionalized particles may be used, e.g., particles that have been aminated, carboxlyated, etc.

LIST OF REFERENCE SIGNS

1: substrate
2: cover
10: flow channel
11: flow through membrane
12: inlet
13: outlet
14: main inlet
15: main outlet
16: auxiliary inlet
17: auxiliary outlet
18: total flow
19: partial flow
20: array of pillars
21: supporting row
22: backflush row
23: pillar
24: supporting row gap
25: backflush row gap
30: membrane
40: bioburden
41: flow rate sensor
42: pressure sensor
43: electronic circuitry
50: EIS control hardware
51: contact pad
61-66: Method steps
71-79: Method steps
WE: working electrode
CE: counter electrode
RE: reference electrode
F: flow direction
w: width of flow channel
h: height of flow channel
g1: supporting row gap width
g2: backflush row gap width
d: distance between supporting row and backflush row

Claims

A microfluidic device comprising:
a flow channel (10) defining a flow direction (F);

an array (20) of pillars (23) arranged in the flow channel (10), the array (20) comprising a supporting row (21) of pillars, the pillars of the supporting row (21) being separated by supporting row gaps (24) having a supporting row gap width (g ₁); and

a membrane (30) comprising colloidal particles (31; 32; 33) which are packed against the supporting row (21) upstream of the supporting row (21) relative to the flow direction (F), at least some of the colloidal particles (31; 32; 33) being larger than the supporting row gap width (g ₁).
The microfluidic device of claim 1, wherein the colloidal particles (31; 32; 33) in the membrane (30) are at least partially fused, the membrane preferably comprising at least 40% by number of colloidal particles that comprise a thermoplastic polymer, in particular, polystyrene.
The microfluidic device of claim 1 or 2,
wherein the array (20) further comprises a backflush row (22) of pillars, the backflush row (22) being arranged upstream of the supporting row (21) at a predefined distance (d) with respect to the flow direction (F),

wherein the pillars of the backflush row (22) are separated by backflush row gaps (25) having a backflush row gap width (g ₂) that is larger than the supporting row gap width (g ₁), and

wherein the membrane (30) extends at least from the supporting row (21) to the backflush row (22).
The microfluidic device of any one of the preceding claims,
wherein at least 20% by number of the colloidal particles (31; 32; 33) in the membrane (30) have a size that is larger than the supporting row gap width (g ₁), preferably by at least 5%.
The microfluidic device of any one of the preceding claims, comprising:
a working electrode (WE) arranged to interact with the membrane (30);

a counter electrode (CE); and

optionally, a reference electrode (RE),

wherein preferably the membrane (30) comprises electrically conductive particles that cause at least a portion of the membrane (30) to be electrically conductive.
The microfluidic device of any one of the preceding claims, wherein the membrane (30) comprises particles that have been functionalized to cause specific binding of at least one target species to the functionalized particles and/or fluorescence in the presence of at least one target species.
The microfluidic device of any one of the preceding claims, comprising an inlet port (12) for supplying a fluid to the flow channel (10) and an outlet port (13) for removing the fluid from the flow channel (10), wherein the membrane (30) is arranged in the flow channel (10) in such manner that the fluid that enters the flow channel (10) through the inlet port (12) traverses the membrane (30) before it leaves the flow channel (10) through the outlet port (13).
The microfluidic device of any one of claims 1-6, comprising:
a main inlet port (14) for supplying a fluid to the flow channel (10) during operation of the microfluidic device;

a main outlet port (15) for removing a first portion of the fluid from the flow channel (10) during operation, the first portion having flown past the membrane (30) without traversing the membrane (30);

an auxiliary outlet port (17) downstream of the membrane for removing a second portion of the supplied fluid from the flow channel (10), the second portion having traversed the membrane (30); and

optionally, an auxiliary inlet port (16) for supplying fluid to the flow channel (10) during assembly of the membrane (30).
The microfluidic device of any one of the preceding claims, wherein the microfluidic device is configured as an exchangeable cartridge.
A bioburden detection system comprising:
the microfluidic device of any one of the preceding claims;

a flow rate sensor (40) for determining a flow rate through the membrane (30) and/or a differential pressure sensor (41) for determining a pressure difference across the membrane; and

optionally, electronic circuitry (42) to determine changes of flow resistance through the membrane due to particle deposition on the membrane, based on the flow rate and/or pressure difference.
A bioburden detection system comprising:
the microfluidic device of claim 5; and

electronic circuitry (50) for carrying out electric impedance measurements, in particular, electrochemical impedance spectroscopy, using the working electrode (WE), counter electrode (CE) and, optionally, reference electrode (RE) of the microfluidic device to determine impedance changes in the microfluidic device due to particle deposition on the membrane (30).
A method of manufacturing a microfluidic device, in particular, the microfluidic device of any one of the preceding claims, the method comprising:
providing a microfluidic chip comprising a flow channel (10) defining a flow direction (F) and an array (20) of pillars (23) arranged in the flow channel (20), the array (20) comprising a supporting row (21) of pillars, the pillars of the supporting row (21) being separated by supporting row gaps (24) having a supporting row gap width (g ₁),

creating a flow of colloidal particles (31; 32; 33) dispersed in a carrier fluid through the flow channel (10) along the flow direction (F), at least some of the colloidal particles (31; 32; 33) being larger than the supporting row gap width (g ₁), to cause flow-directed assembly of a membrane (30) that comprises the colloidal particles (31; 32; 33); and

sintering the membrane (30) to at least partially fuse the colloidal particles (31; 32; 33) in the membrane (30).
The method of claim 12, comprising:
during or after assembly of the membrane and before sintering, applying a flow rate ramp to increase a packing density of the colloidal particles (31; 32; 33) in the membrane (30).
The method of claim 12 or 13, comprising functionalizing at least some of the colloidal particles.
A method of detecting bioburden using the microfluidic device of any one of claims 1-9, comprising:
causing a flow of a fluid through the microfluidic device; and

monitoring a flow resistance through the membrane and/or an electrical impedance in the microfluidic device and/or fluorescence at the membrane to detect the bioburden.