WO2022243366A1

WO2022243366A1 - Centrifugal microfluidic device

Info

Publication number: WO2022243366A1
Application number: PCT/EP2022/063427
Authority: WO
Inventors: Jeppe FOCK; Mattias PARMVI; Martin GEDSTED CHRISTENSEN; Robert BABER; Shou ZANG; John Mccormack
Original assignee: Blusense Diagnostics Aps
Priority date: 2021-05-20
Filing date: 2022-05-18
Publication date: 2022-11-24

Abstract

A centrifugal microfluidic device comprising a plurality of inlets for receiving fluids, and a plurality of distinct detection chambers. The device comprises: a first inlet for receiving a fluid, the first inlet provided in connection to at least one detection chamber, a second inlet for receiving a fluid sample, the second inlet provided in connection to at least one detection chamber, a third inlet for receiving a fluid sample, the third inlet provided in connection to at least one detection chamber, wherein the second inlet is fluidically connected to the first inlet via a fluidic path such that a fluid provided to the second inlet may interact with a fluid provided to the first inlet, and wherein the third inlet is fluidically sealed during operation of the device from the first inlet such that a fluid provided to the third inlet may not interact with the fluid provided to the first inlet.

Description

CENTRIFUGAL MICROFLUIDIC DEVICE

Field of the Invention

The present disclosure relates to centrifugal microfluidic devices. In particular it relates to centrifugal microfluidic devices comprising a plurality of inlets and detection chambers.

Background of the invention

Microfluidic devices are for the processing and handling liquids in the nanolitre to microlitre range. Microfluidic devices comprise fluidic chambers for receiving and processing, retaining, and/or holding liquids, and channels for the delivery and routing of liquids throughout the device. Centrifugal microfluidic devices are a type of microfluidic device whereby centrifugal forces are used to pump fluids through the device.

Centrifugal microfluidic devices are rotated such that a centrifugal force acts upon the liquid in the device. Centrifugal microfluidic devices may comprise passive valves which use the surface tension of the liquid within the channels and chambers to create capillary stops, sometimes described as fluidic seals or closures, generally at regions whereby the channel geometry is altered, such as rapidly widened. The device is rotated to generate a centrifugal force sufficient to break such capillary stops and in such a manner the flow of fluid through the device can be sequenced and time-controlled.

The flow of fluid through the microfluidic device when the device is not rotating may be achieved in different manners. Siphons that prime based on capillary action are one such manner. However, such siphons may not be sufficiently reliable, or efficient on their own. This is especially so when the surface tension of the fluids may vary, impacting the rate of capillary wicking in the siphons. Improved devices and techniques for the flow of fluids in passive siphons would be advantageous. Additionally, active valves are known in the art. However, active valves introduce complexity as external activation of the valves is required.

Centrifugal microfluidics is especially used in the life sciences, in particular in lab-based analytics and diagnostics. As operations such as pipetting, mixing, measuring, aliquoting and centrifuging are possible to automate via centrifugal microfluidics it is especially relevant in environments where such operations may be difficult to control such as small-scale or remote laboratories lacking traditional and expensive analytic devices.

Generally centrifugal microfluidic devices comprise a single inlet for receiving a fluid sample, and a plurality of chambers and channels. As an example of a centrifugal microfluidic device, EP 2 028496 B1 (Samsung Electronics Co. Ltd.) describes a device comprising a single specimen chamber and inlet for receiving a sample/specimen. The single specimen chamber and inlet is connected to a plurality of dilution chambers which are pre-filled with a volume of diluent. The device comprises a plurality of reaction chambers which may form detection chambers. However, the device of EP 2 028496 B1 is for receiving a single specimen which is diluted to different degrees on-chip via the pre-filled dilution chambers and then reacted in the reaction chamber(s). In ‘496 device the device accepts a fluid via the single sample inlet each of the reaction chambers receives a diluted portion of the sample in all configurations. The ‘496 device further requires active wax valves to control fluid transfer. Centrifugal microfluidic devices are ideal for diagnostics due to their small sample volume requirements, reaction times, and their size. The capability of detecting multiple analytes from a single sample and thereby providing improved diagnostic reliability would be ideal. Additionally, the provision of a device which enables multiple varied protocols on the same device platform would be ideal. Such a platform enabling multiple protocols lowers the total cost of production and negates the need the need for different device layouts for each protocol.

Summary of the invention

Accordingly, the present invention preferably seeks to mitigate, alleviate or eliminate one or more of the above-identified deficiencies in the art and disadvantages singly or in any combination and solves at least the above mentioned problems by providing a centrifugal microfluidic device comprising a plurality of inlets for receiving fluids, and a plurality of distinct detection chambers. The device comprises: a first inlet for receiving a fluid, the first inlet provided in connection to at least one detection chamber, a second inlet for receiving a fluid sample, the second inlet provided in connection to at least one detection chamber, a third inlet for receiving a fluid sample, the third inlet provided in connection to at least one detection chamber, wherein the second inlet is fluidically connected to the first inlet via a fluidic path such that a fluid provided to the second inlet may interact with a fluid provided to the first inlet, and wherein the third inlet is fluidically sealed during operation of the device from the first inlet such that a fluid provided to the third inlet may not interact with the fluid provided to the first inlet.

Further advantageous embodiments are disclosed in the appended and dependent patent claims.

Brief description of the drawings

These and other aspects, features and advantages of which the invention is capable will be apparent and elucidated from the following description of embodiments of the present invention, reference being made to the accompanying drawings, in which:

Fig. 1 is a schematic top-down view of a centrifugal device according to an aspect.

Fig. 2a is a schematic top-down detail view of a part of a centrifugal device comprising a first vented chamber, and a second chamber which is non-vented due to the presence of liquid in the first chamber.

Fig. 2b is a schematic top-down detail view of a part of a centrifugal device comprising a first vented chamber, and a second chamber in a vented state due to the presence of liquid in the second chamber.

Fig. 2c is a schematic top-down detail view of a part of a centrifugal device comprising a first vented chamber, a second chamber, and a third chamber each in a vented state where liquid is present in various elements of the structure.

Detailed description

Figure 1 shows a centrifugal microfluidic device 1 comprising a plurality of inlets 101, 102, 103 for receiving fluids. The inlets 101, 102, 103 are fluidically connected as will be described below to detection chamber(s) 104, 105, 106 for detecting or analysing a fluid sample.

The first inlet 101 is fluidically connected to the second inlet 102, via a fluidic path such that a fluid provided to the first inlet 101 may mix or otherwise interact with a fluid provided to the second inlet 102. The fluidic path may comprise chambers 108a, 108b, 110, 112, channels 109, 111 and/or fluidic valves 113, 114 which control and guide the fluid(s) provided to the first or second inlets 101, 102.

The third inlet 103 is fluidically connected to at least one detection chamber 104. The third inlet 103 is connected to at least one detection chamber 104 via a fluidic path which during use is fluidically disconnected from the first inlet 101, such that a fluid provided to the third inlet 103 cannot interact with a fluid provided to the first inlet 101. The third inlet 103 is advantageously connected, during use, to the detection chamber 104.

The first inlet 101 is for receiving a fluid comprising a diluent. The first inlet 101 is fluidically connected to at least two of the plurality of detection chambers 105, 106. Generally, the first inlet 101 is for receiving a diluent or a pre-diluted fluid sample. Fluid provided to the first inlet 101 flows via a transfer channel to a metering chamber 108a,b. The metering chamber 108a,b may be provided with a first metering portion 108a, for metering a volume of fluid, and a second portion 108b for receiving an excess volume of fluid which exceeds the capacity of the metering portion 108a. Fluid in the metering chamber 108a,b may be transferred from the metering chamber 108a,b to a dilution chamber 116 via a fluidic path such as a channel 109 provided with a siphon 114 as shown in figure 1. As will be described below, the first inlet 101 is not connected to the detection chamber 104 which is connected to the third inlet 103.

The fluidic path from the first inlet 101 to the at least one detection chamber (105, 106) is provided with at least one passive valve configured to control the flow of fluid during operation of the device. Such passive valves are described further below.

The first inlet 101 may be radially inward of the second inlet 102, and the third inlet 103.

As opposed to existing devices, the device 1 is not pre-filled with a diluent. This enables the provision of different volumes of diluent, and fluid samples comprising a diluent depending on specific testing protocol for a sample or samples.

The second inlet 102 is for receiving a fluid sample. The second inlet 102 may be for receiving a non-diluted physiological sample. The second inlet 102 may be for receiving a prediluted fluid sample in some aspects or uses of the device. The fluid provided to the second inlet 102 is subsequently receivable in at least one detection chamber 104, 105, 106. The second inlet 102 may be fluidically connected to at least two, such as each of, of the detection chambers 104, 105, 106. That is, fluid provided to the second inlet 102 may be receivable in each of the detection chambers 104, 105, 106. The second inlet 102 may coincide with, that is be provided to a chamber 110, for receiving the fluid sample. The second inlet 102 is in fluidic connection with the dilution chamber 116. The fluidic path between the second inlet 102, and the dilution chamber 116 may comprise fluidic channels 111, 115 and/or a sample metering chamber 112a,b for metering a volume of the fluid sample provided to the second inlet 102. The fluidic path between the second inlet 102 and the plurality of detection chambers 104, 105, 106 may be provided with at least one passive valve configured to control the flow of fluid during operation of the device. For example, a siphon or other passive fluidic valve 113 may be provided on the fluidic path between the second inlet 102 and the dilution chamber 116 such that the flow of fluid from the second inlet 102 to the dilution chamber 116 may be controlled.

The second inlet 102 may be radially outward of the first inlet 101. The second inlet 102 may be radially inward of the first inlet 101.

The third inlet 103 is for receiving a fluid sample. The third inlet 103 may receive a fluid sample comprising a diluent. That is, a fluid comprising a physiological sample and a diluent, for example, a pre-diluted fluid sample. The fluid sample provided to the third inlet 103, need not comprise a diluent in all aspects or uses of the device. The fluid sample provided to the third inlet 103 may be a non-diluted fluid sample. The fluid sample provided to the third inlet is subsequently receivable in at least one detection chamber 104. The third inlet 103 is connected to at least one detection chamber 104 via a fluidic path. The fluidic path between the third inlet 103 and the detection chamber 104 is sealed during operation of the device from the fluidic path connecting the first inlet 101, and the second inlet 102 to the detection chambers 105, 106. Sealed as used here means that the fluid provided to the third inlet 103 is substantially restricted from flowing to the fluidic path connecting the first inlet 101 and the second inlet 102 to the detection chambers 105, 106. The restriction may be achieved via at least one fluidic valve, such changes in the geometry of channels provided between the fluidic path connecting the first and second inlets 101, 102 to the detection chambers 104, 105, 106, and the fluidic path connecting the third inlet 103, to the detection chamber 104.

The third inlet 103 may be radially outward of the first inlet 101, and the second inlet 102. The radial location of the respective inlets 101, 102, 103 combined with the passive valves as described herein enables the reliable transfer of liquid within the device 1

As stated, the flow of fluid from the third inlet 103, toward the inlets 101, 102 is restricted, or sealed during use/operation of the device. That is, flow is allowed in the direction from the third inlet 103, to the detection chamber 104, however, flow of fluid from the third inlet 103 toward the first 101, or second 102 inlets, and the detection chambers 105, 106 is restricted.

As can be seen in figure 1, the third inlet 103 may coincide with a chamber 117 for receiving the fluid provided to the third inlet 103. The chamber 117 may be referred to as the bypass chamber 117 as fluid provided to the third inlet 103 bypasses the flow path in connection with the first inlet 101, and second inlet 102 and continues to the detection chamber 104 without passing via the dilution chamber 116.

The first, second and third inlets 101, 102, 103 as described above may also be referred to as sample injection ports, sample ports, or injection ports as is known in the art. The first, second and third inlets 101, 102, 103 are openings such that they may receive a sample via a device for the provision of a sample, such as a pipette, dropper etc. As opposed to vents which are dimensioned such that they enable the expulsion of air from a fluidic structure, the inlets 101, 102, 103 are configured to receive a liquid.

As described above, the first inlet 101 is fluidically connected to at least two of the plurality of detection chambers 105, 106. The second inlet 102 is fluidically connected to each of the plurality of detection chambers 104, 105, 106. The third inlet 103 is provided in fluidic connection to one detection chamber 104, and not fluidically connected during use to the detection chambers 105, 106 in fluidic connection to the first inlet 101.

The inlets 101, 102, 103 and their respective connections to detection chambers 104, 105, 106 enable a single device 1 which can receive different types of fluids depending on a specific testing protocol. For example, in a first example test protocol a non-diluted physiological fluid sample may be provided to the second inlet 102 and a diluent may be provided to the first inlet 101. The non-diluted sample is metered and provided to the dilution chamber 116, and separately to the detection chamber 104. The diluent is metered and then provided to the dilution chamber 116 to be mixed with the non-diluted sample. The diluted physiological sample may subsequently flow to the detection chamber(s) 105, 106.

In a second test protocol, a diluted physiological sample, that is a physiological fluid sample comprising a diluent may be provided to the first inlet 101, the diluted physiological sample is metered and then subsequently provided to detection chamber(s) 105, 106.

In a third test protocol, a physiological sample may be provided to the third inlet 103, the sample does not flow to the dilution chamber 116. The sample provided to the third inlet may subsequently flow to the detection chamber 104. The sample provided to the third inlet 103 in the third test protocol described may be a pre-diluted sample, that is a physiological sample comprising a diluent.

The above protocols may be advantageously combined depending on the available sample types. For example, a first physiological fluid sample may be provided to the first inlet 101, and a second physiological fluid sample may be provided to the third inlet 103. The first physiological sample may subsequently flow to at least one of the detection chambers 105, 106, such as two detection chambers 105, 106. A second physiological sample may be provided to the third inlet 103. The second physiological sample may flow to at least one of the detection chambers 104, that is where the detection chamber is not the detection chamber which receives the first sample, the first and second physiological samples may thereby be simultaneously analysed on a single device. The first and second samples need not be the same type of physiological sample.

In the manner described above, a single device 1 is capable of receiving different sample types, both un-diluted and/or pre-diluted depending on the test protocol without any change in structure of the device 1. This enables a device 1 which can be used in various point-of-care, laboratory diagnostic, or other settings for various sample types. Referring again to figure 1, the various elements will now be described in detail. Inlet 101 is provided in fluidic communication with a metering chamber 108a, b. Fluid provided to the first inlet 101 may be received in a chamber 118 for initial storage and receiving the fluid provided to the inlet 101. Fluid received into the chamber 118 may then flow into the metering chamber 108a,b via the channel connecting the chamber 118 to the metering chamber 108a, b. The capacity of the chamber 118, and the fluid provided at the inlet 101, may exceed the volume of the first portion 108a of the metering chamber 108a,b, excess fluid provided to the inlet 101 is received in the second portion 108b of the metering chamber 108a,b. The metering chamber 108a,b may be vented to enable the fluid in the metering chamber 108a,b to enable the flow of fluid into and from the metering chamber 108a, b.

Under application of an external force, generally rotation of the device 1, fluid in the metering chamber 108 flows into the channel 109 comprising the siphon 114. Once the capillary forces exceed the external forces, the siphon will become primed and subsequent external forces will move the volume of fluid in the first portion 108a of the metering chamber 108a,b into the dilution chamber 116.

As can be seen in figure 1, the dilution chamber 116 may be vented at a radially inward portion. From the dilution chamber 116 it is possible to distribute the fluid to the one, several of, or the plurality of detection chambers 105, 106. In the device shown in figure 1, two of the detection chambers 105, 106 are fluidically connected to the dilution chamber 116. In the device of figure 1, fluid cannot flow from the dilution chamber 116 to the detection chamber 104. However, as stated previously, fluid may flow from the second inlet 102, or the third inlet 103 to the detection chamber 104, however this fluid does not pass via the dilution chamber 116. In such an arrangement, a portion of the fluid may be diluted, mixed or otherwise processed in the dilution chamber 116, whilst a second portion may be transferred to the detection chamber 104, without interaction with the fluid from the first inlet 101.

The dilution chamber 116 may be provided with a channel 119 at a radial outward portion of the chamber 116 for distribution of the fluid from the dilution chamber 116 to various chambers, such as, for example the detection chambers 105, 106. In the device shown in figure 1, the channel 119 connects the dilution chamber to two of the detection chambers 105, 106. The microfluidic device 1 may be provided with a chamber and channel arrangement 120, 123, 124, 125 adapted to alter from a non-vented to a vented state via the displacement of fluid. In particular, a chamber 124 may be altered from a non-vented to a vented state via the provision of liquid to the chamber 124. The arrangement is shown in as part of the device in figure 1 and separately in detailed figures 2a and b. In figures 2a and 2b, liquid is shown as the waved region. The arrangement comprises a first chamber 120 having an inlet 1201 for the provision of fluid, a first vented outlet 1202 for venting gas/air, and a second outlet for fluid 1231. A second chamber 124 is connected downstream of the first chamber 120. A channel 125 is connected at both its inlet and outlet to the second chamber 124. Fluid provided via the inlet 1201 will remain in the chamber 120 due to the provision of a fluidic stop at the second outlet 1231 and as the structures downstream of the first chamber 120, for example chambers 124 and channel 125 are closed to atmosphere. Below a specific threshold force, e.g., rotation force, fluid will not flow into the downstream structures 124, 125 due to the opposing forces of the air compressing within the structures downstream of the first chamber 120. Liquid remaining in chamber 120 is shown in figure 2a.

Above a specific threshold transfer force, for example, by rotating the device above a specific rate, the fluid in the first chamber 120 is transferred completely to the second chamber 124. The second chamber 124 has an outlet 1251 to the channel 125 at a radially outward portion. The second chamber 124 has an inlet 1252 from the channel 125 at a radially inward portion. As can be seen in figures 1 and 2, the channel 125 is connected at both ends to the chamber 124. The channel 125 is a sealed channel from the radial outward portion of the chamber 124 to the radial inward portion of the chamber 124. The outlet 1251 from the chamber 124 may be considered the inlet 1251 to the channel 125. The inlet 1252 to the chamber 124 may be considered the outlet 1252 of the channel 125.

A siphon valve 1253 is provided to the channel 125. The siphon valve 1253 has a crest which is radially inward of the inlet 1251 of the channel 125. The siphon valve 1253 is radially outward of the outlet 1252 of the channel 125. The crest of the siphon valve 1253 is radially outward of the first chamber 120. The crest of the first siphon valve 1253 is radially outward of the second outlet 1231. After transfer of the fluid from the first chamber 120 to the second chamber 124 the second chamber 124 is vented to atmosphere via the vent 1202 provided to the first chamber 120. To achieve this the volumetric capacity of the second chamber 124, and a portion of the channel 125 between the inlet 1251 and the siphon valve 1253 must be such that a space is present at the radial inward portion of the chamber 124 enabling air to vent via channel 123 and chamber 120 to the vent 1202. The volumetric capacity of the second chamber 124 and the portion of the channel 125 between the inlet 1251 and the siphon valve 1253 is greater than the volume of fluid provided to the first chamber 120. Figure 2b shows clearly the liquid level in the chamber 124 and the region, radially inward of the liquid where air can vent to the vent 1202 via the chamber 120.

The second chamber 124 is fluidically connected to a third chamber 105. As shown in figures 1 and 2, the channel 125 is provided with the third chamber 105. The second chamber 124 is connected to the third chamber 105 via the siphon valve 1253. As the third chamber 105 is provided on the channel 125 the third chamber 105 is vented to atmosphere via the outlet 1252 and the vent 1202 provided to the chamber 120. Fluid primes the siphon valve 1253 when capillary forces exceed centrifugal forces which thereafter displaces air from the channel 125, and the third chamber 105, back via the outlet of channel 125 to the first chamber 120 and to the vent 1202.

The channel 125 is provided with a second portion 1254. The second portion is downstream of the first siphon valve 1253. The second portion is downstream of the third chamber 105. The second portion 1254 is extends from the third chamber 105 to the outlet 1252 and has a crest radially inward of the first siphon valve 1253.

The arrangement of chambers 120, 124 and 105 allows, as will be elaborated below, for example resuspension and/or mixing of a suspension, emulsion etc. The fluid is maintained in the first chamber 120 when the applied external force is below the threshold force. The fluid may be incubated or maintained for a duration whilst chemical binding events or other reactions occur.

Additionally, the arrangement of chambers 120, 124 and first and second siphon valves 1253, 1254 enables a fluidic structure where fluid is maintainable in the second chamber 124 in transitory states and does not tend to flow back upstream as the volume of fluid is chamber 124 is non-pressurised. Additionally, fluid is maintainable in the chamber 124 at variable device rotation rates. Once the full volume of fluid has entered chamber 124 there are no pressurised volumes in the downstream structure as the arrangement is vented via 1202. Furthermore, as will be described below fluid in chamber 124 may undergo a separation stage, subsequently be provided to the third chamber 105, and thereafter be maintained in the third chamber 105 without the requirement of constant higher rotation speeds as the third chamber 105 is effectively vented via the vent 1202.

The second chamber 124 may be provided with two portions 124a, 124b as shown in figure 1 and 2. The second portion 124b may be for trapping a solid phase, pellet, sediment etc. The second portion 124b is radially outward of the first potion 124a. The first potion 124a is radially inward of the second portion 124b. The channel 125 is connected to the first portion 124a. The inlet 1251 to the channel 125 is connected at a radially outward portion of the first portion 124a. The outlet 1252 of the channel 125 is connected to a radially inward portion of the first portion 124a of the second chamber 124. If the chamber comprises the second portion 124b then the second portion is radially outward of the inlet to the channel 125. The fluid, or supernatant if the fluid has undergone separation via the two portions 124a, 124b, is transferred via the channel 125 to the third chamber 105.

In some cases, separation of a particles from a supernatant may not be necessary, and in such a case the second chamber 124 need not comprise the second portion 124b.

As shown in figure 1, the fluidic structure enabling venting after transfer of fluid from a first chamber 120 to a second chamber 124 may be provided between the dilution chamber 116 and at least one of the detection chambers 105 connected to the dilution chamber 116. In such an arrangement the detection chamber 105 forms the third chamber described above. The first chamber 120 is a reaction chamber for removing at least one molecule present in the sample. The chamber 120 for removing at least one molecule present in the sample may be provided with an agent 121, such as a solid affinity resin 121 for capturing the at least one molecule. For example, a solid affinity matrix 121 may be provided in the chamber 120. The agent 121 may be selected such that a molecule present in the sample is bound to the agent 121. Advantageously, the agent 121 is a resin or plurality of particles which mixes with the fluid provided to the chamber 120. The agent 121 may be received in an element 122, such as a recess 122 for holding the agent prior to the introduction of fluid to the chamber 120. When the agent 121 mixes with the fluid the agent 121 binds the molecule to be bound. For example, the agent 121 may be a solid affinity resin which is selected to bind with an antibody such as human immunoglobulin G (IgG) and thereby any human IgG present in the fluid is bound to the particles. The agent 121 may bind a portion, ideally a majority of the non-target molecule in the sample. The reaction/mixing time of the fluid in the chamber 120 is controlled via the venting structure described above. The mixture of agent 121 and fluid may thereafter flow into the chamber 124 for receiving the mixture. Advantageously, as described above the chamber 124 is provided with a first portion 124a for receiving the mixture comprising suspended particles and a liquid, and a second portion 124b for receiving sedimented particles. The second portion 124b is radially outward of the first portion 124a. To ensure that the sedimented particles are maintained in the second portion 124b, the geometry of the chamber 124 is such that the chamber 124 has a narrow region between the two portions 124a, 124b. The channel 125 is connected at a radial outward portion of the first portion 124a of the chamber 124 and connected at a radial inward portion of the first portion 124a of the chamber 124. That is, both ends of the channel 125 are connected to the chamber 124. Supernatant in 124a flows into the channel 125 at the inlet provided at a radial outward portion of the first portion 124a of the chamber 124. The inlet 1251 to the channel 125 is provided radially inward of the second portion 124b of the chamber 124 to ensure that the sedimented particles are maintained in the second portion 124b and the supernatant in 124 a may selectively flow into the channel 125.

The detection chamber 105 is provided at a point along the channel 125. The detection chamber 105 is radially outward of the inlet 1251 to the channel 125 and the outlet 1252 of the channel to the chamber 125. At a point on the channel 125, between the inlet to the channel 125 and the detection chamber 105 a mixing chamber 126 may be provided. The mixing chamber 126 may comprise detection particles selected to bind to a specific target molecule in a fluid sample. The detection particles bound to the target molecule in the fluid sample may then flow into the detection chamber 105. The device 1 may then be subjected to a detection process to detect the concentration/amount of target molecule in the fluid sample in the detection chamber 105. By providing the detection particles in the mixing chamber 126 the duration of mixing of the fluid sample with the detection particles may be better controllable than if the detection particles were provided in the detection chamber 105 alone. Furthermore, the mixing chamber 126 may be provided with an agent selected to bind or block to a non-target molecule in the sample. The agent, which may be considered a blocking agent, may be selected to bind to the same non-target molecules bound to the affinity resin the chamber 120, for example to bind to a protein present in the sample, such as, IgG. In such an arrangement, the amount of blocking agent required in the mixing chamber 126 is less than would be required without the affinity resin being present in the chamber 120 as a substantial proportion of the non-target molecule has previously been removed from the sample via immobilisation and sedimentation on binding particles.

The detection particles and/or the blocking agent may be provided in the detection chamber 105 in some instances. In such an arrangement, the mixing chamber 126 may not need to be provided to the channel 125. However, a combination whereby the blocking agent and/or the detection particles are provided to the mixing chamber 126 and the detection particles/blocking agent are provided to the detection chamber 105 is also possible to implement in the present device 1.

The detection particles may be functionalised magnetic nanoparticles. A biosensor for the detection of analytes using functionalized magnetic nanoparticles is described inEP 3 014245 Bl. InEP 3 014245 B1 the magnetic nanoparticles which have been functionalized with bioactive ligands is described. The magnetic nanoparticles may be functionalised with immobilised antigens. The antigens bind to antibodies in the fluid sample and cause the agglutination of magnetic nanoparticles which may be enhanced by the application of a magnetic field supplied by a reader device 2.

The flow path of fluid from the dilution chamber 116 to the detection chamber 105 has been described. As can be seen in figure 1, a fluid in the dilution chamber 116 may also flow into channel 119 and subsequently into to detection chamber 106. A portion of fluid from the dilution chamber 116 may flow subsequently to detection chamber 105, and second portion of the fluid may flow from the dilution chamber 116 to detection chamber 106. The portion of fluid which flows to detection chamber 106 does not flow via the chamber 120, and therefore may comprise the non-target molecule which is advantageously blocked via sedimentation in chamber 120.

Such that the fluid flow from the dilution chamber 116 to the detection chamber 105 and to detection chamber 106 may be selectively controlled, the fluidic path between the dilution chamber 116 and the detection chambers 105, 106 may be provided with a chamber 127. The chamber 127 has an outlet at a radially inward portion for distribution of the fluid to the detection chamber 105, and an outlet at a radially outward portion for distribution of the fluid to the detection chamber 106. The geometry and position of the two outlets enable the selective flow of fluid to the detection chamber 105, and to the detection chamber 106. That is, the volume of fluid preferentially flows via the first radially outward outlet toward the chamber 106, and subsequently to detection chamber

105 via the chamber 120. The chamber 127 further enables additional metering of the sample. As with the fluidic path from the chamber 120 to the detection chamber 105, the channel 128 may be provided with a mixing chamber 129. The mixing chamber 129 may comprise detection particles selected to bind to a specific target molecule in a fluid sample, or a plurality of target molecules in the sample, or target molecules and non target molecules in the sample. The detection particles bound to the target molecules in the fluid sample may then flow into the detection chamber 106. The device 1 may then be subjected to a detection process to detect the amount of target molecule in the fluid sample in the detection chamber 106.

The channel 128 at which the detection chamber 106 is positioned has a vent 134 at a point downstream from the detection chamber 106. Both detection chambers 105,

106 are therefore positioned on non-dead-end channels. The detection chambers 105, 106 may be referred to as being open, that is they are not closed/dead-end chambers without an outlet. As has been described above, the detection chamber 105 is vented via the chamber 120 and vent 1202 only when 120 is empty from liquid. In such an arrangement fluid flows into the detection chamber 105, 106 after the provision of an initial force, to e.g., break any fluidic seals at the inlets/outlets of the respective channels 125, 128, and flows without further actuation into the detection chambers 105, 106. This means that the device does not need to be continuously rotated at high speed to force fluid into the detection chambers 105, 106.

As described previously, a fluid provided to the second inlet 102 may flow via a flow path to the dilution chamber 116 and subsequently to detection chambers 105, 106. The fluid provided to the second inlet 102 may flow into the sample metering chamber 112. The sample metering chamber 112 may be provided with a radially inward portion 112a and a radially outward portion 112b, the two portions being separated by a narrow region. The two portions enable the metering chamber 112 to also fractionate for example, a fluid sample comprising red blood cells (RBCs). The RBCs may be fractionated from a whole blood sample such that RBCs are captured in the radially outward portion 112b and substantially blood plasma remains in the radially inward portion 112a. Any excess sample volume may flow into an overflow chamber 131.

As described previously, a fluid provided to the third inlet 103 may flow via a flow path to the detection chamber 104. The fluid provided to the third inlet 103 will not interact with a fluid provided to the first inlet 101. In particular, a fluid provided to the third inlet 103 will not enter the dilution chamber 116, and therefore, will not enter detection chambers 105, 106. The flow path between the third inlet 103 and the detection chamber 104 may be provided with a chamber 130 for metering and/or fractionation of the fluid sample. As with the chamber 112, the chamber 130 may be provided with a radially inward portion 130a and a radially outward portion 130b separated by a narrow region. The chamber 130 may act as a combined fractionation and metering chamber whereby the excess fluid volume exceeding the capacity of the chamber 130 flows into the overflow chamber 131.

As shown in figure 1, both the second inlet 102, and the third inlet 103 are connected to the chamber 130. The fluidic connection between the second inlet 102 and the chamber 130 is such that fluid generally may flow only from the inlet 102 to the chamber 130, and not from the chamber 130 to the second inlet 102. In such an arrangement, fluid cannot flow from the third inlet 103, via the chamber 130, to the fluidic path in connection with the second inlet 102.

The chamber 130 is connected to the detection chamber 104 by channel 132. Channel 132 may be provided with a chamber for reacting the portion of the sample provided to the second 102 or third inlet 103 metered from the metering chamber 130. The reaction may for example be a blocking step whereby a non-target molecule in the sample is blocked or otherwise reacted such that it is less likely to be detected in the detection chamber 104. As can be seen in figure 1, the channel 132 between the third inlet 103 and the detection chamber 104 may be provided with a mixing chamber 133. The mixing chamber 133 may comprise detection particles selected to bind to a specific target molecule in a fluid sample, or a plurality of target molecules in the sample, or target molecules and non-target molecules in the sample. The detection particles bound to the target and or non-target and target molecules in the fluid sample may then flow into the detection chamber 104. The device 1 may then be subjected to a detection process to detect the concentration/amount of target molecule in the fluid sample in the detection chamber 104. A chamber 135 may be provided upstream of the detection chamber 104, and when present, upstream of the mixing chamber 133. The chamber 135 may form a chamber for storing fluid, whereby a fluidic seal is formed at a radial outward position such that the fluid to be provided to the detection chamber 104 may be maintained in the chamber prior to rotation at a rate such that fluid in chamber 135 is forced into the detection chamber 104.

The detection chamber 104 is a dead-end chamber without a vent to enable the escape of air in the chamber 104. The detection chamber 104 may be referred to as closed. This means that the device 1 must be rotated to force fluid to enter the chamber 104. This is as opposed to the detection chambers 105, 106 which do not require the provision of a rotation force exceeding a burst pressure once fluid has entered chambers 124 and 127 respectively. This enables selective detection via detection chamber 104 based on the rotation speed of the device 1.

As described above, when detecting analytes, it may be desirable to block, immobilise and/or precipitate specific components from the sample such that they do not interfere with the detection of target analyte(s) and measurement processes. For example, it may be desirable to block, immobilise and/or precipitate a specific antibody from the sample such that it will not interfere with a detection process for another antibody. Generally, the sample may comprise, for each detection process, a target molecule, i.e., an analyte and at least one non-target molecule. The non-target molecule may otherwise impact the detection and measurement if it is present during the detection process. For example, the non-target molecule may bind non-specifically to a detection particle. A blocking, immobilisation, and/or precipitation process is a process whereby non-target molecules are selectively blocked, immobilised and/or precipitated such that the target molecule remains in solution. A medium for blocking, immobilisation, and/or precipitation may comprise a specific blocking molecule selected to react with a non target molecule.

Within the device 1 blocking, immobilisation and/or precipitation may be performed in the chamber 120 for removing at least one molecule present in the sample. In chamber 120 immobilisation is performed by binding a portion of the sample, such as a protein, to the agent 121. Mixing the sample with the agent 121 causes a proportion, such as a majority of the non-target molecule to be bound to the agent 121 such that it cannot bind or otherwise interfere with the detection process. A proportion, such as a majority of the non-target molecule may be captured by the agent 121. The liquid in chamber 120 may be partially or substantially free of non-immobilised non-target molecule. In practice, it is likely that the liquid comprises a small amount of non-target molecule which was not captured by the agent 121. To ensure that a greater portion of the non-target molecule will not interfere with the detection process an additional blocking process may be performed in mixing chamber 126.

In a typical example, the agent 121 binds to IgG present in the sample and a majority of the IgG in the sample is bound to the agent and subsequently captured, sedimented, and flows into the second portion 124b of chamber 124, the second portion 124b receives and captures the sediment such that it does not flow downstream into the detection chamber 105.

The mixing chamber 126 may advantageously be provided with an agent selected to block and/or immobilise a second molecule in the sample. To block and/or immobilise this second molecule an agent is provided to the mixing chamber 126 which is selected to bind to a molecule present in the sample that was not blocked/bound to the agent 121. The agent in the mixing chamber 126 may be a solid affinity matrix for binding to the non-target molecules in the sample. The agent in the mixing chamber 126 may be a reagent such as a buffer comprising anti-human immunoglobulin A or immunoglobulin G antibodies (anti-IgA or anti-IgG), or a combination, that will form complexes on mixing with the IgA present in the supernatant and thereafter not bind to the detection particles present in the mixing chamber 126. As described above, the mixing chamber 126 may comprise the detection particles in addition to any agents adapted to block and/or immobilise non-target molecules.

The mixing chamber 126 may also be provided with a blocking agent to block and/or immobilise the same non-target molecule blocked by the agent 121 in the chamber 120. That is, in the above example with IgG, a separate and additional IgG blocking agent may be present in the mixing chamber 126. As the majority of IgG has already been removed by the agent 121, the amount of blocking agent required in the mixing chamber for the same non-target molecule as the agent 121 blocks is reduced. This two-step process advantageously reduces the amount of blocking agent required in the mixing chamber 126 and thereby reduces the risk that any complexes or sediment formed in the mixing chamber 126 adversely affects the signal detectable in the detection chamber 105. This two-step process, in addition to the blocking, immobilisation, and/or precipitation performed in chamber 120, reduces the problems associated with typical immunoprecipitation as described previously.

As would be understood the sample entering the detection chamber 105 has, according to the above procedure been processed such that a first non-target molecule has been removed/reduced via the agent 121, and the first and/or second non-target molecule has been removed/reduced via the blocking agent present in the mixing chamber 126. The target molecule may then bind to detection particles in the mixing chamber 126 and subsequently be detected in the detection chamber 105. In the above procedure, if the detection particles are selected to bind to immunoglobulin M (IgM) then the sample in the detection chamber 105 will comprise detection particles bound to IgM and the total amount of IgM in the sample will be better detectable due to the step-wise removal of non-target molecules in the proceeding chambers 126, 120. As would be apparent this arrangement could be used to detect one of several target molecules after removal of non target molecules form the sample. That is, it need not be human IgM which is detected in detection chamber 105, but could be any target molecule, and the non-target molecules need not be IgG/IgA, but could be any non-target molecule which could bind to the detection particles in the mixing chamber 126, and potentially disturb the detection process in the detection chamber 105.

Importantly, for the device 1, the detection chambers generally detect different molecules in the sample. That is, the target molecule, and the non-target molecules are different for each of the detection chambers 104, 105, 106. In the proceeding paragraphs the detection of IgM in the detection chamber 105 was described, whereby IgG and IgA are blocked/removed from the sample prior to IgM detection. Thereby, the detected amount of IgM is more accurate as there is reduced risk of non-specific binding within the detection chamber 105.

The detection chamber 106 may detect a total concentration of molecules including target and non-target molecules. This may be considered a total concentration. Using the example of human antibody detection again, the detection chamber 106 may detect each of IgG, IgA and IgM or Total Antibody. That is, whereas the detection chamber 105 detects the amount of a specific target molecule in a sample, detection chamber 106 may detect a total amount of analytes, including analytes which are removed from the sample in detection chamber 105.

As a specific example, the detection particles in the mixing chamber 129 may be selected to bind to more than one human antibody type, such as each of IgG, IgM and IgA.

As the sample which mixes with the detection particles in chamber 129, has not been blocked by the agent 121 in the chamber 120, nor has it encountered the additional blocking agent in mixing chamber 126, it continues to comprise each of the target and non-target molecules. The total amount of target and non-target molecule present in the sample detected in chamber 106 may be compared to the specific amount of target molecule present in the sample detected in chamber 105. For example, the specific amount of IgM may be compared to the total antibody amount enabling more accurate diagnosis of the sample based on relative antibody amounts.

In the device 1 shown in figure 1, detection chambers 105, 106 may detect antibody amounts, detection chamber 104 may advantageously detect an antigen. In such an arrangement the device 1 is adapted to detect both an acute infection and serological (antibody) response to an infection/disease. The device 1 may be considered to comprise two portions. A first portion A comprising a detection chamber 104, and a second portion B comprising at least two detection chambers 105, 106. In figure 1, the two portions A, B are shown on either side of the dotted line. The first portion A is for detecting a first target molecule. The second portion B is for detecting both a second target molecule, and a non-target molecule. The second portion B does not detect the first target molecule.

The first detection portion A comprises the detection particles which bind to the first target molecule. The second detection portion B comprises the detection particles which bind to the second target and non-target molecules.

As is described above, the control of fluid sample in the device 1 is achieved via passive valves. The device 1, and specifically neither the first nor the second detection portions A, B comprise an active valve. The transfer of fluid sample within the first detection portion A to the first detection chamber 104 is controlled by at least one passive valve. The transfer of fluid sample within the second detection portion B to the second detection chamber 105 is controlled by at last one passive valve. The transfer of fluid sample within the second detection portion B to the third detection chamber 106 is controlled by at least one passive valve. As is described above, the detection particles may be provided to two separate and distinct mixing chambers 126, 129. The provision of the fluid sample to the mixing chambers 126, 129 is selectable via respective passive valves. As the device lacks complex active valves, the provision of fluid to the first detection portion A and to the second detection portion is controlled exclusively by controlling the rotation speed of the device 1. This simplifies operation of the device, and simplifies the system with which the device is used as it need not comprise additional means for activating and controlling active valves.

The first target molecule may be an antigen. The second target molecule may be an antibody, for example, IgM or IgA. The non-target molecule may be an antibody, for example IgG.

As described above, the device l is a centrifugal microfluidic device. To actively flow fluid through the channels and chambers of the microfluidic device 1 the device is generally rotated. The microfluidic device 1 is rotated around a central rotational axis. The central rotational axis is perpendicular to the general plane of fluid flow in the microfluidic device 1. As the device 1 is rotated when in use, the device 1 may be considered to comprise a radial inner edge 10 and a radial outer edge 11. Portions or features of the device closer the radial inner edge 10 are considered radially inner portions or features. Portions or features which are closer to the radial outer edge 11 are considered radial outer portions or features. A radial outer feature is proximal the outer radial edge 11, distal the inner radial edge 10. A radial inner feature is proximal the radial inner edge 10, distal the radial outer edge 11. The radial inner edge 10 is proximal the central rotational axis. The radial outer edge 11 is distal the central rotational axis. If an element or feature is described as being radially inward, or internal, of a second element, that refers to the first element’s position being proximal the inner edge 10 with respect to the second element. If an element of feature is described as being radially external, or outward, of a second element, that refers to the first element’s position being proximal the outer edge 11 with respect to the second element. The terms downstream and upstream relate to the relative location of elements. A first element is upstream of a second element if fluid first flows to the first element, and then to the second element. Fluid generally flows from a radial inward portion of the device outwards, due to the applied rotational force. However, this need not always be the case, as for example, as described throughout the application, various channels may comprise siphons, which are a fluidic valve element which directs a channel radially inward, over a crest, and then radially outward. Upstream and downstream elements are clearly determinable to the skilled person via comparing the location of the named elements in the figures.

As opposed to existing devices, the present device 1 does not comprise active valves. Active valves are valves that can be switched on or off by external means. For example, wax valves have previously been used within centrifugal microfluidic devices to control the provision of fluids at different time points. However, the provision of active valves increases the total complexity of the system as for example, heating means are required to activate the valves. The flow of fluid within the present device 1 is controlled by passive valves such as siphons, capillary and hydrophobic valves, centrifugal- pneumatic valves and does therefore not require active valves.

When the device 1 is not rotated the fluid in the device will settle or will flow due to other forces than centrifugal force, such as capillary forces. Such that the detection chambers 104, 105, 106 can be used with a light-based detection system, at least the base of the detection chambers 104, 105, 106 is substantially transparent to light in the visible and ultraviolet wavelengths. Light is directed to the base and/or the top of the detection chambers 104, 105, 106 and is transmitted through the base and/or top of the detection chambers 104, 105, 106 to, for example, magnetic nanoparticles therein. An aperture may be provided above, or below the detection chambers 10, 105, 106 coaxial with the centre of each respective chamber. The aperture may have at least one angled wall with respect to the Z plane (into the page in Figs 1 and 2) such that incident light not directed at the radial centre of the detection chamber 104, 105, 106 is reflected away and is not transmitted to the magnetic nanoparticles. This may improve the quality of the detected signal as only sample in the centre of the respective detection chamber 104, 105, 106 receives light.

The microfluidic device 1 comprises a substrate and a cover. As would be understood the fluidic structures such as channels, chambers, vents etc. may be provided in either the substrate, the cover, or both. The fluidics structures are substantially within the same plane, however, certain fluidic structures may be in, for example, the cover, and certain fluidic structures may be, for example in the substrate, leading to them being in different, but adjacent planes.

Some, such as a plurality of channels and chambers of the device 1 may be provided with a pre-wetting agent to improve the flow of a fluid through the device 1. The channel 109 may be provided with a pre-wetting agent. The agent may be provided downstream of the siphon 114. The channel 115 may be provided with a pre-wetting agent downstream of the siphon 113. The channel 132 may be provided with a pre-wetting agent upstream of the mixing chamber 133 and/or the storage chamber 135. The channel 119 may be provided with a pre-wetting agent at a position upstream of the chamber 127. Each of the mixing chambers 126, 129, 133 may be provided with a pre-wetting agent. The provision of the pre-wetting agent enables improved control of the flow of fluid through the device 1.

The detection particles, the pre-wetting agents, and the agent 121 may each be provided to the device 1, prior to assembly. That is, in a device comprising a substrate and a cover they may be provided to the channels and chambers before the provision of the cover. This ensures that the operation of the device 1 is simpler for an end-user as no pre-treatment steps are required and the device 1 may be used via the provision of a fluid sample.

The microfluidic device 1 comprises structures in the micrometre range such that volumes of liquids in the nanolitre to microlitre range may be processed on the device 1. The term fluid used herein is used in the sense of a microfluidic device and therefore refers generally to a liquid fluid. If a gas fluid is meant, such as air, then it is referred to as a gas.

The device 1 may be provided with channels or portions of chambers having an irregular indent 208, 230, 231. In particular, the portion of the metering chamber 108 connecting the first portion 108a to the second portion 108b may be provided with an irregular indent 208. The irregular indent 208 may be referred to as a fillet, or hook in the channel/chamber wall. The indent 208 reduces the risk of unintentional siphoning from the first portion 108a to the second portion 108b, from the metered portion 108a to the overflow portion 108b. With a continuous flat channel/chamber wall the inventors have identified that there is a risk of a capillary forming along the wall. The indent 208 limits capillary formation along channel/chamber edges. Furthermore, if a capillary is formed, the indent 208 reduces the height of siphons formed by inverting the edge where the siphon forms in the direction opposite the pseudo-gravitation forces driving flow radially outward. A similar indent 230 may be provided to the channel connected the sample metering chamber 112 to the metering chamber 130. Furthermore, an indent 231 may be provided between the metering chamber 130 and the overflow chamber 131.

The microfluidic device 1 may be formed from any suitable material, such as a glass or plastic. Suitable plastics may for example be PMMA, PC, PS, COC, or PDMS etc. A microfluidic assembly may comprise a plurality of microfluidic devices 1 such that an array of tests may be run simultaneously.

Generally, the fluid sample comprises a biological substance. The sample may comprise whole blood, serum, plasma, mucous, urine, or a combination thereof. The sample may be mixed with a buffer, diluent or reagent as is known in the art. Although, the present invention has been described above with reference to specific embodiments, it is not intended to be limited to the specific form set forth herein. Rather, the invention is limited only by the accompanying claims.

In the claims, the term “comprises/comprising” does not exclude the presence of other elements or steps. Additionally, although individual features may be included in different claims, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and/or advantageous. In addition, singular references do not exclude a plurality. The terms “a”, “an”, “first”, “second” etc do not preclude a plurality. Reference signs in the claims are provided merely as a clarifying example and shall not be construed as limiting the scope of the claims in any way.

Claims

1. A centrifugal microfluidic device (1) comprising a plurality of inlets (101, 102, 103) for receiving fluids, and a plurality of distinct detection chambers (104, 105, 106), wherein the device (1) comprises:

-a first inlet (101) for receiving a fluid, the first inlet (101) provided in connection to at least one detection chamber (105, 106),

-a second inlet (102) for receiving a fluid sample, the second inlet (102) provided in connection to at least one detection chamber (104, 105, 106),

-a third inlet (103) for receiving a fluid sample, the third inlet (103) provided in connection to at least one detection chamber (104), wherein the second inlet (102) is fluidically connected to the first inlet (101) via a fluidic path such that a fluid provided to the second inlet (102) may interact with a fluid provided to the first inlet (101), and wherein the third inlet (103) is fluidically sealed during operation of the device from the first inlet (101) such that a fluid provided to the third inlet (103) may not interact with the fluid provided to the first inlet (101).

2 The centrifugal microfluidic device (1) according to claim 1, wherein the first inlet (101) and the second inlet (102) are provided in fluidic connection to a chamber (116), and wherein the chamber (116) is provided in fluidic connection to at least one detection chamber (105, 106).

3. The centrifugal microfluidic device (1) according to claim 1 or 2, wherein the first inlet (101) is fluidically connected to at least two of the plurality of detection chambers (105, 106).

4. The centrifugal microfluidic device according to any of claims 1 to 3, wherein the second inlet (102) is fluidically connected to each of the plurality of detection chambers (104, 105, 106).

5 The centrifugal microfluidic device (1) according to any of claims 1 to 4, wherein the third inlet (103) is provided in fluidic connection to one detection chamber (104), and not fluidically connected during use to the detection chambers (105, 106) being in fluidic connection to the first inlet (101).

6. The centrifugal microfluidic device (1) according to any of claims 1 to 5, wherein the third inlet (103) is fluidically connected to a metering chamber (130), and wherein, the metering chamber (130) is fluidically connected to the second inlet (102),

7. The centrifugal microfluidic device (1) according to claim 6, wherein the fluidic connection between the second inlet (102) and the metering chamber (130) is such that fluid cannot flow from the metering chamber (130) toward the second inlet (102) during use of the device (1).

8 The centrifugal microfluidic device (1) according to any of claims 1 to 7, wherein the first inlet (101) is for receiving a fluid comprising a diluent.

9. The centrifugal microfluidic device (1) according to any of claims 1 to 8, wherein the second inlet (102) is for receiving a physiological fluid sample.

10. The centrifugal microfluidic device (1) according to any of claims 1 to 9, wherein the third inlet (103) is for receiving a physiological fluid sample.

11 The centrifugal microfluidic device (1) according to any of claims 1 to 10, wherein the fluidic path between the first inlet (101) and the at least one detection chamber (105, 106) is provided with at least one passive valve configured to control the flow of fluid during operation of the device (1).

12. The centrifugal microfluidic device (1) according to any of claims 1 to 11, wherein the fluidic path between the second inlet (102) and the plurality of detection chambers (104, 105, 106) is provided with at least one passive valve configured to control the flow of fluid during operation of the device (1).

13. The centrifugal microfluidic device (1) according to any of claims 1 to 12, wherein the fluidic path between the third inlet (103) and the at least one detection chamber (104) is provided with a passive valve to control the flow of fluid during operation of the device (1).

14. The centrifugal microfluidic device (1) according to any of claims 1 to 13, wherein the device (1) does not comprise an active valve.

15. The centrifugal microfluidic device (1) according to any of claims 1 to 14, wherein the device (1) is not pre-filled with a diluent.

16. The centrifugal microfluidic device (1) according to any of claims 1 to 15, wherein the second inlet (102) is radially inward of the third inlet (103).

17. The centrifugal microfluidic device (1) according to any of claims 1 to 16, wherein the third inlet (103) is radially outward of the first inlet (101) and the second inlet (102).