WO2017151195A1

WO2017151195A1 - Nucleic acid molecular diagnosis

Info

Publication number: WO2017151195A1
Application number: PCT/US2016/063372
Authority: WO
Inventors: Weihua Guan; Gihoon CHOI
Original assignee: The Penn State Research Foundation
Priority date: 2016-02-29
Filing date: 2016-11-22
Publication date: 2017-09-08

Abstract

The present invention provides cartridge devices and disk-based systems for portable molecular diagnoses and methods for using the same. While the invention is disk-based, it represents an improvement over the prior art by manipulating liquids without relying on centripetal force. The devices and systems require less power to operate, are less mechanically complicated, and occupy a smaller space. The devices and systems are useful for performing molecular diagnoses in the field, where portability, reliability, and device stamina are of particular importance.

Description

TITLE

NUCLEIC ACID MOLECULAR DIAGNOSIS

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62/395,170 filed September 15, 2016, and to U.S. Provisional Patent Application No. 62/301,408 filed February 29, 2016, the contents of which are each incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with government support under Grant No.

UL1TR000127, awarded by the National Institutes of Health/National Center for

Advancing Translational Sciences. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Molecular diagnostic tests detect specific sequences in DNA or RNA associated with a specific disease. Molecular diagnosis is widely used in a wide range of clinical areas such as infectious diseases, oncology, pharmacogenomics, genetic disease screening, human leukocyte antigen typing, and coagulation. The general nucleic acid based molecular diagnostic assay requires three basic steps: (1) Sample preparation - extraction and purification of interested nucleic acid; (2) Amplification - making copies of the nucleic acid of interest (target); and (3) Results reading - detection of the amplified target. Molecular diagnosis requires specialized infrastructure for preparing high quality molecule samples, highly skilled technicians, electricity, and expertise to interpret the results. Many of these requirements are not often readily available outside centralized facilities.

The need for rapid and accurate molecular diagnoses in the field has led to developments in point-of-care devices that attempt to incorporate every aspect of nucleic acid based molecular diagnosis. For example, the prior art has attempted to create disc- based point-of-care devices for diagnosis in the field (Yoo et al., WO 2014/104830; Kinahan et al., WO 2014/198939). While these devices may possess the ability to perform molecular diagnoses, their dependence on centripetal acceleration to manipulate fluids leads to substantial power consumption and limits portability.

There is a need in the art for an improved devices, systems, and methods for point-of-care diagnosis. The present invention meets this need.

SUMMARY OF THE INVENTION

The present invention relates to cartridge devices and disk-based systems for portable molecular diagnoses and methods for using the same. In one aspect, the invention relates to a cartridge device for point-of-care molecular diagnoses comprising: a substantially planar casing; at least one chamber within the casing; at least one microchannel connecting the at least one chamber within the casing; and at least one inlet in the casing providing access to the at least one chamber; wherein the at least one chamber and at least one microchannel are aligned in an arc.

In one embodiment, the at least one chamber includes a lysing chamber, a binding chamber, a washing chamber, and a reaction chamber. In one embodiment, the lysing chamber is preloaded with a volume of lysis buffer. In one embodiment, the binding chamber is preloaded with a volume of binding buffer with pH-switchable magnetic beads, wherein the binding buffer comprises a pH below 6.5. In one embodiment, the washing chamber is preloaded with a volume of washing buffer. In one embodiment, the reaction chamber is preloaded with a volume of LAMP reaction mix, wherein the LAMP reaction mix comprises a pH above 8.8. In one embodiment, the LAMP reaction mix further comprises a fluorescent dye. In one embodiment, the at least one microchannel comprises a capillary valve for separating the contents of the at least one chamber. In one embodiment, the at least one microchannel comprises a

hydrophobic coating for separating the contents of the at least one chamber. In one embodiment, the arc comprises a geometry proportional to a segment of the

circumference of a disk in a disk-based cartridge reading system. In one embodiment, the device is disposable. In another aspect, the invention relates to a disk-based cartridge reading system, comprising: a disc comprising at least one cartridge slot; at least one substantially planar cartridge comprising at least one chamber and at least one microchannel connecting the at least one chamber, wherein the at least one chamber and at least one microchannel are aligned in an arc; a servomotor; at least one magnet; at least one heating element; at least one photodetector; and at least one light source; wherein the disk is capable of holding at least one cartridge, and wherein the servomotor is capable of actuating the disk to position the chambers of the cartridge near the at least one magnet, the at least one heating element, the at least photodetector, and the at least one light source.

In one embodiment, the at least one magnet is an electromagnet. In one embodiment, the at least one light source is one of a laser or an LED. In one

embodiment, the system further comprises a component selected from the group consisting of: a GPS component, a digital display component, a portable battery power component, a wireless transmitting and receiving component, a microcontroller component, and a data reading and writing component.

In another aspect, the invention relates to a method of point-of-care molecular diagnosis. The method comprises the steps of: acquiring a sample; acquiring a cartridge comprising a lysing chamber with lysing buffer, a binding chamber with binding buffer and magnetic beads, a washing chamber with wash buffer, and a reaction chamber with LAMP reaction mix, wherein the chambers are connected in sequence by a plurality of microchannels; placing the sample in the lysing chamber with lysing buffer to release sample nucleic acids; placing the cartridge in a disk-based cartridge reading system comprising a disk, at least one magnet, at least one heating element, at least one photodetector, and at least one light source; activating the at least one magnet and actuating the disk such that the magnetic beads and binding buffer are drawn into the lysing chamber by the at least one magnet, wherein the magnetic beads bind the sample nucleic acids; actuating the disk such that the magnetic beads and sample nucleic acids are drawn into the buffer chamber, the washing chamber, and the reaction chamber by the at least one magnet; deactivating the magnet and actuating the disk to position the reaction chamber near the at least one heating element to carry out a LAMP reaction; and actuating the disk to position the reaction chamber near the at least one photodetector and the at least one light source to detect fluorescence, wherein the detection of fluorescence indicates a positive diagnosis.

In one embodiment, an amount of 10 to 100 μL of sample is acquired. In one embodiment, the sample is pre-lysed prior to placement in the lysing chamber of the cartridge. In one embodiment, the actuation timing and actuation speed of the disk are adjusted based on the type of diagnosis being performed. In one embodiment, the heating element maintains a temperature of 60° C for the duration of the LAMP reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figure 1 depicts an exemplary cartridge design of the present invention.

Figure 2 depicts an exemplary prototype cartridge of the present invention.

Figure 3A through Figure 3C depicts an illustration of an exemplary reagent compact disc and integrated sample preparation on the compact disc. (Figure 3 A) Exploded view of the reagent compact disc showing three patterned PMMA layers.

(Figure 3B) Assembled view of the reagent compact disc showing three independent testing units. Each test unit consists of five chambers: a DNA binding chamber (binding buffer pH 5.0), a washing chamber (washing buffer pH 7.0), a reaction chamber (master mix, pH 8.8), and two valving chambers. All reagents are preloaded on the compact disc in a ready-to-use format. The lysate was prepared by collecting 10 μΕ malaria-infected blood into 1 mL of lysis buffer in a microcentrifuge tube. (Figure 3C) Illustration of integrated sample preparation and amplification steps on the compact disc. By rotating the compact disc against a stationary magnet in a specifically designed control sequence (steps 1-4), the pH charge switchable magnetic beads were directed from chamber to chamber, which allows for seamlessly integrated DNA binding, purification, elution, and amplification on the compact disc.

Figure 4A and Figure 4B depict an overview of an exemplary standalone and mobile nucleic acid testing system. (Figure 4A) Schematic diagram of the assembled system with the reagent compact disc. The whole platform is of a small footprint (12x13x13 cm). The reagent compact disc was secured on the rotatable spindle platter. (Figure 4B) Schematic diagram of functional parts in an exploded view.

Figure 5 depicts the design of an exemplary disk, cartridge, and internal system components. The accompanying flowchart illustrates the sample analysis steps capable of being performed by the system.

Figure 6 depicts a block diagram of the system modules. The platform consists of four main functional modules: mechanical modules (servo motor/spindle platter/compact disc), optical modules (LED/optical sensor), thermal modules (Peltier heater/thermal sensor), and data connectivity modules (Bluetooth). Each module was controlled by a microprocessor on a customized PCB board. The diagnostic results can be optionally reported to a smartphone user interface.

Figure 7 depicts a flowchart illustrating an exemplary method of using the disk-based cartridge reading system of the present invention.

Figure 8 depicts the results of an experiment demonstrating the viability of using a loop mediated isothermal amplification (LAMP) assay using lab-extracted DNA samples from three standard isolates (Pf529, 3D7, and D10) of Plasmodium falciparum species and negative controls. The emitted fluorescence indicates a positive reaction. The excitation is by a 480nm blue LED.

Figure 9 depicts the feedback-controlled reaction temperature profiles as a function of time, (blue curve: system 1, red curve: system 2).

Figure 10A depicts an illustration of the manual parasite genomic DNA extraction and purification procedures in a microcentrifuge tube. Figure 10B depicts the amplification curve for the manually extracted DNA sample on the instrument. The successful amplification of the tube-extracted DNA samples validates the effectiveness of the magnetic bead-based method.

Figure 1 1 A depicts the reagent setup of an exemplary LAMP master mix. Figure 1 IB depicts a P. falciparum specific primer set.

Figure 12 depicts the location of LAMP target sequence and priming sites of Plasmodium falciparum (Pf: Genbank accession no. AJ276844). The core priming sites of inner/outer primers (F3/B3, F2/B2, and F lc/B lc) with additional priming sites of loop primers (LF/LB) are marked on the sequence.

Figure 13 A and Figure 13B depict the determination of the amplification threshold time (Tt). (Figure 13 A) A real-time amplification curve. (Figure 13B) The differential profile of the real-time amplification curve (dRFU/dt), the max of which is used to determine the amplification threshold time. (Tt: threshold time, t: time, Mmax: maximum value of the slope).

Figure 14 depicts the steps for operating the system from sample to answer. 10 μL of the whole blood sample was collected into 1 mL of lysis buffer by using the capillary tube and the lysate was ready to be loaded into the compact disc after incubation at room temperature for 2 minutes (steps 1-3). 180 μL lysate was introduced to the compact disc through the loading inlet which was then sealed by pressure-sensitive adhesive tape (step 4). The sealed compact disc was inserted into the instrument and the system can be optionally connected to a smartphone user interface (step 5-6). When closing the lid, the system automatically performs all assay steps including nucleic acid purification, elution, amplification and real-time detection (step 7). The diagnostic result can be reported within 40 min on an LCD screen or optionally on the smartphone user interface (step 8).

Figure 15A and Figure 15B depict illustrations of the pinning effect and photo images of the drop test results. (Figure 15 A) A droplet on a solid surface with a contact angle of 6>, which will be increased up to θ+α when moving towards a three- phase edge, where a is a bending angle (Enayati, A. et al., Annual Review of

Entomology, 2010, 55 :569-591). This implies that larger a allows a higher activation barrier for the passive valve. (Figure 15B) The drop test to evaluate the robustness of the teeth-shaped passive valves on the reagent compact disc under the harsh mechanical vibration. (N denotes the number of drops).

Figure 16A through Figure 16D depicts the results of validating the LAMP assay and the instrument with laboratory purified DNA samples. (Figure 16 A) Benchmarking amplification curves obtained from the real-time PCR machine, lx, O. lx, and O.Olx denotes the dilution factors of the P. falciparum DNA samples (NTC: No template controls, Tt: Threshold time). (Figure 16B) The amplification profiles acquired from the instrument. (Figure 16C) Gel-electrophoresis analysis on a 2% agarose gel. The amplicons show a clear ladder-like pattern, the length of which verifies the LAMP assay's specificity against P. falciparum. (Figure 16D) Emission visualized under the blue LED (λ=488 nm) illumination for various positive and negative samples.

Figure 17 depicts the results of validating the system from the whole blood sample to the amplification result with integrated sample preparation on the compact disc. The % value represents the parasitemia of the infected RBCs. (hRBC: healthy RBCs, NTC: No template controls, T_t: Threshold time).

Figure 18 depicts the sensitivity of the system for detecting P. falciparum infected blood samples. Amplification curves for various infected blood samples of different parasitemia. The inversely proportional relationship between the amplification threshold time and the parasitemia confirms the quantitative ability of the system. A detection limit of -0.6 parasites^L (i.e., 0.00002% parasitemia) against P. falciparum was successfully achieved.

Figure 19 depicts the cost breakdown for an exemplary instrument.

Figure 20 depicts the disposable reagent compact disc cost per test.

Figure 21 depicts the DNA amplification profiles of the system. The manufacturing of the instrument is repeatable in a cost-effective way. A second instrument was built and a similar sensitivity experiment was performed (as described in the sensitivity section). Amplification curves from six different parasitemia samples show clear exponential increases of fluorescence, while that of the negative controls (master mix and hRBC) shows no amplification. (RFU: relative fluorescence unit, hRBC: healthy RBCs, NC: negative control). DETAILED DESCRIPTION

Definitions

It is to be understood that the figures and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for the purpose of clarity, many other elements typically found in the art. Those of ordinary skill in the art may recognize that other elements and/or steps are desirable and/or required in implementing the present invention. However, because such elements and steps are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements and steps is not provided herein. The disclosure herein is directed to all such variations and modifications to such elements and methods known to those skilled in the art.

Unless defined elsewhere, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

As used herein, each of the following terms has the meaning associated with it in this section. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

"About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%), ±1%), and ±0.1% from the specified value, as such variations are

appropriate.

Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, 6, and any whole and partial increments there between. This applies regardless of the breadth of the range.

Cartridge

In one aspect, the present invention provides cartridges for use in discbased systems for point-of-care molecular diagnoses. While disc-based diagnosis systems exist in the art (e.g., Yoo et al., WO 2014/104830; Kinahan et al., WO

2014/198939), the art is dependent on centripetal acceleration to manipulate fluids. The power and mechanisms needed to drive the disks to rates in excess of 600-2000 rpm results in bulky systems prone to malfunction. Furthermore, the disk systems in the prior art use very sophisticated and error-prone valves to control the separation, splitting, metering, and combining of reagents, which demands a large area and limits the number of samples that can be run at a time. In contrast, the present cartridges provide a simple, linear chamber design that does not rely on centripetal acceleration to manipulate fluids, leading to lower power consumption and a smaller footprint. Referring now to Figure 1, the layout of an exemplary cartridge 10 is depicted. Cartridge 10 comprises at least lysing chamber 12, binding chamber 14, washing chamber 17, and reaction chamber 18. In some embodiments, cartridge 10 may comprise additional chambers as needed. For example, in Figure 2, cartridge 10 further comprises one or more valving chambers 16. Valving chambers 16 provide additional spacing between chambers containing reagents. In various embodiments, valving chambers 16 can be filled with any suitable material, such as air or an oil. The chambers of cartridge 10 are connected in sequence by a plurality of microchannels 20. In certain embodiments, microchannels 20 are treated to be hydrophobic, such that any fluids within the chambers stay separated. In some embodiments, microchannels 20 are at least partially sealed by capillary valves 21. In certain embodiments, microchannels 20 comprise pointed, teeth-shaped tips, wherein the tips provide a partial seal through liquid surface tension. Referring now to Figure 2, a prototype cartridge 10 is depicted. The chambers and microchannels of cartridge 10 are aligned in an arc, wherein the arc geometry is proportional to a segment of the circumference of a disk in a disk-based cartridge reading system described elsewhere herein.

The chambers and microchannels of cartridge 10 are housed within a substantially planar casing 32, wherein casing 32 comprises at least one inlet 34 for inserting samples and liquids. Casing 32 can comprise any suitable material, such as plastics or glass. Casing 32 is preferably at least partially transparent. In some embodiments, casing 32 is disposable.

In certain embodiments, the chambers and microchannels of cartridge 10 are incorporated into a disc. Referring now to Figure 3 A through Figure 3C, an exemplary disc housing 33 is depicted. Disc housing 33 can contain any suitable number of chambers and microchannels. For example, the equivalent of one, two, three, or more cartridges 10 can be retained within disc housing 33. Disc housing 33 can comprise any suitable material, such as plastics or glass. Disc housing 33 is preferably at least partially transparent. In some embodiments, disc housing 33 is disposable.

In some embodiments, cartridge 10 is provided with all chambers empty. Cartridge 10 may be loaded with one or more reagents prior to use by inserting the one or more reagents through the at least one inlet 34. In other embodiments, cartridge 10 is provided with at least one chamber preloaded with a reagent. For example, lysing chamber 12 may be preloaded with lysis buffer 22; binding chamber 14 may be preloaded with binding buffer 24 and a plurality of magnetic beads 26; washing chamber 28 may be preloaded with washing buffer 28; and reaction chamber 18 may be preloaded with LAMP reaction mix 30. In some embodiments, the chambers are preloaded with 25 to 100 μΐ_^ of their respective reagents.

Lysis buffer 22 can be any lysis buffer suitable for lysing a sample to release nucleic acids. For example, a lysis buffer may comprise reagents such as Tris-Cl, EDTA, Tween 20, Triton X-100, and the like. Magnetic beads 26 are preferable pH- switchable, wherein the beads comprise a coating that is reversibly ionizable based on pH, such as the ChargeSwitch magnetic beads. For example, magnetic beads 26 may comprise a positive charge at low pH and a neutral charge at high pH. Binding buffer 24 can be any suitable buffer having a pH below 6.5, such that magnetic beads 26 in binding buffer 24 comprise a positive charge to attract negatively charged nucleic acids. Washing buffer 28 can be any suitable wash buffer compatible with an ionizable magnetic bead system, such as a wash buffer having a pH below 6.5 to maintain the positive charge of magnetic beads 26.

LAMP reaction mix 30 can be any suitable LAMP reaction mix. As will be understood by those having skill in the art, typical LAMP reaction mixes include forward inner primers (FIP), backward inner primers (BIP), F3/B3 primers, and reagents such as Tris-HCl, (NH₄)₂S0₄, KC1, MgS0₄, Tween 20, Betaine, MnCl₂, dNTPs, and Bst DNA Polymerase. In some embodiments, LAMP reaction mix 30 comprises a fluorescent dye, such as calcein. In one embodiment, an exemplary 25 μΐ_^ amount of LAMP reaction mix 30 comprises: 1.6 μΜ each of FIP/BIP primers, 0.8 μΜ each of LF/ LB primers, 0.2 μΜ each of F3/B3 primers, 20mM Tris-HCl, lOmM ( H₄)₂S0₄, 50mM KC1, 8mM MgS0₄, 0.1% Tween 20, 0.8 M Betaine, 25 μΜ of Calcein, 0.75 mM MnCh, 1.4 mM dNTPs, 0.32 ωιίί/μί Bst DNA Polymerase. Preferably, the pH of LAMP reaction mix 30 is above 6.5, such as a pH of 8.8, to render the magnetic beads 26 neutrally charged and to elute nucleic acids into the LAMP reaction mix. Disk-Based Cartridge Reading System

In another aspect, the present invention provides disk-based cartridge reading systems for molecular diagnoses. The systems accept cartridge 10 of the present invention and comprise components to manipulate the samples and reagents within cartridge 10. As described elsewhere herein, the systems do not rely on centripetal acceleration to manipulate fluids, leading to lower power consumption and a smaller footprint.

Referring now to Figure 4A and Figure 4B, an exemplary cartridge reading system 40 is depicted. While cartridge reading system 40 may comprise a plurality of shapes and sizes, typical embodiments comprise a body 42, lid 44, and cartridge disk 46. The internal components include, but are not limited to, optical excitation component 58, optical detecting component 56, magnet 52, heating plate 54, and servo motor 50. Body 42 and lid 44 can comprise any suitable material, including plastics and metals. Body 42 and lid 44 house the internal components of cartridge reading system 40 in a portable, rigid shell.

Referring now to Figure 5, an exemplary layout of the internal components of cartridge reading system 40 is depicted. Disk 46 comprises at least one cartridge slot 48 for accepting cartridge 10. Servomotor system 50 is provided to actuate and rotate disk 46. Positioned at various locations around servomotor system 50 are at least one magnet 52, at least one heating element 54, at least one photodetector 56, and at least one light source 58 (not pictured). The at least one magnet 52 is preferably an electromagnet, such that magnetism can be activated and deactivated at will. The at least one heating element 54 can be any heating element capable of maintaining a temperature suitable for sustaining a LAMP reaction. The at least one photodetector 56 can be any suitable photodetector capable of detecting fluorescence. The at least one light source 58 can be any suitable light source capable of fluorescence excitation, such as a laser or LED light. Each of the aforementioned components are positioned such that when disk 46 is actuated, the chambers of a cartridge 10 attached to disk 46 may be positioned above, or at least near, any one of the aforementioned components. Referring now to Figure 6, an exemplary cartridge reading system 40 design sketch is depicted. Cartridge reading system 40 may comprise additional components such as a digital readout LCD display, a global positioning system, and a means for storing and transferring data. In some embodiments, cartridge reading system 40 may further comprise wireless capabilities, such as Bluetooth, for wirelessly communicating with companion devices to transfer and receive data and input commands. A means of portable power, such as a rechargeable lithium battery, is provided to power cartridge reading system 40.

As contemplated herein, cartridge reading system 40 includes a system platform for controlling the various components. In some embodiments, the system of the present invention may operate on a computer platform, such as a local or remote executable software platform, or as a hosted internet or network program or portal. In certain embodiments, only portions of the system may be computer operated, or in other embodiments, the entire system may be computer operated. As contemplated herein, any computing device as would be understood by those skilled in the art may be used with the system, including desktop or mobile devices, laptops, desktops, tablets, smartphones or other wireless digital/cellular phones, televisions or other thin client devices as would be understood by those skilled in the art. In some embodiments, the computer platform is an Arduino microcontroller. The platform is fully capable of being integrated for use with any data recording, analysis, and output procedures as described herein throughout.

The computer platform is fully capable of actuating the disk-based system and activating the various components for molecular diagnoses as described herein throughout. For example, the computer platform can be configured to control the speed and timing of disk actuation to correspond to sample incubation times in the various cartridge chambers, perform the necessary heating and fluorescence excitation steps, and subsequently transmit the results of fluorescence detection to a digital display. The computer platform may further provide a means to communicate the results, such as by projecting one or more static and moving images on a screen, presenting one or more digital readouts, and the like. The computer operable component(s) of the system may reside entirely on a single computing device, or may reside on a central server and run on any number of end-user devices via a communications network. In one embodiment, the computer operable components reside in at least one Arduino microcontroller. The computing devices may include at least one processor, standard input and output devices, as well as all hardware and software typically found on computing devices for storing data and running programs, and for sending and receiving data over a network, if needed. If a central server is used, it may be one server or, more preferably, a combination of scalable servers, providing functionality as a network mainframe server, a web server, a mail server and central database server, all maintained and managed by an administrator or operator of the system. The computing device(s) may also be connected directly or via a network to remote databases, such as for additional storage backup, and to allow for the communication of files, email, software, and any other data formats between two or more computing devices. There are no limitations to the number, type or connectivity of the databases utilized by the system of the present invention. The communications network can be a wide area network and may be any suitable networked system understood by those having ordinary skill in the art, such as, for example, an open, wide area network (e.g., the internet), an electronic network, an optical network, a wireless network, a physically secure network or virtual private network, and any combinations thereof. The communications network may also include any intermediate nodes, such as gateways, routers, bridges, internet service provider networks, public-switched telephone networks, proxy servers, firewalls, and the like, such that the communications network may be suitable for the transmission of information items and other data throughout the system.

The system software may also include standard reporting mechanisms, such as generating a printable results report, or an electronic results report that can be transmitted to any communicatively connected computing device, such as a generated email message or file attachment. Likewise, particular results of the aforementioned system can trigger an alert signal, such as the generation of an alert email, text or phone call, to alert a manager, expert, researcher, or other professional of the particular results. Further embodiments of such mechanisms are described elsewhere herein or may standard systems understood by those skilled in the art.

Methods of Making

The devices of the present invention can be made using any suitable method known in the art. The method of making may vary depending on the materials used. For example, devices substantially comprising a plastic or polymer may be milled from a larger block or injection molded. Likewise, devices substantially comprising a metal may be milled from a larger block of metal or may be cast from molten metal. In some embodiments, the devices may be made using 3D printing techniques commonly used in the art.

Method of Diagnosis

In another aspect, the present invention provides a method for molecular diagnosis using the devices and systems of the present invention. Referring now to Figure 7, an exemplary method 100 of molecular diagnosis is presented. Method 100 begins with step 110, wherein a sample is acquired. The sample can be any sample containing nucleic acids, such as blood. In some embodiments, a sample volume between 10 and 100 μΐ. is acquired. In step 120, a cartridge is acquired, wherein the cartridge comprises a lysing chamber with lysing buffer, a binding chamber with binding buffer and magnetic beads, a washing chamber with wash buffer, and a reaction chamber with LAMP reaction mix, and wherein the chambers are connected in sequence by a plurality of microchannels. In step 130, the sample is placed in the lysing chamber with lysing buffer to release sample nucleic acids. In step 140, the cartridge is placed in a disk-based cartridge system comprising a disk, at least one magnet, at least one heating element, at least one photodetector, and at least one light source. In step 150, the at least one magnet is activated and the disk is actuated such that the magnetic beads and binding buffer are drawn into the lysing chamber by the at least one magnet, wherein the magnetic beads bind the sample nucleic acids. In step 160, the disk is actuated such that the magnetic beads and sample nucleic acids are drawn into the buffer chamber, the washing chamber, and the reaction chamber by the at least one magnet. In step 170, the at least one magnet is deactivated and the disk is actuated to position the reaction chamber near the at least one heating element to carry out a LAMP reaction. In some embodiments, the heating element maintains a temperature of 60° C near the reaction chamber. In step 180, the disk is actuated to position the reaction chamber near the at least one photodetector and the at least one light source to detect fluorescence, wherein the detection of fluorescence indicates a positive diagnosis.

As described elsewhere herein, the present invention does not use any centripetal force to manipulate fluids. Rather, as depicted in method 100, sample nucleic acid and fluids are manipulated through the use of magnetic beads and magnets. After sample acquisition, the sample is placed in the lysing chamber to release sample nucleic acids. In some embodiments, the sample may be lysed separately from a cartridge. For example, a sample may be placed in lysing buffer in a container separate from the cartridge, and the pre-lysed sample is placed in the cartridge.

When the cartridge is placed in the disk-based cartridge reading system, the disk aligns the chambers of the cartridge with the at least one magnet, at least one heating element, at least one photodetector, and at least one light source for subsequent sample treatment. Fluid and sample manipulation occurs first when the disk is actuated to position the magnetic beads near the magnets and the magnets are activated. After magnet activation, the magnetic beads remain spatially locked above the magnets. The disk may then be actuated to relocate a chamber of the cartridge above the magnets and contain the spatially locked magnetic beads and any surrounding liquid. At any point, the magnets may be deactivated to release the magnetic beads and any surrounding liquid in its most current chamber, whereby actuating the disk will relocate the chamber and magnetic beads together.

Accordingly, the sample nucleic acids may be released in the lysing chamber and relocated in sequence to the binding chamber, washing chamber, and reaction chamber by activation of the magnets, and subsequently subjected to heat treatment from the at least one heating element, then fluorescence excitation and detection from the at least one light source and the at least one photodetector by deactivation of the magnets. Actuation parameters pertaining to timing and speed may be adjusted to control incubation times in any chamber.

In some embodiments, step 150 through step 180 may be performed automatically. For example, an operator need only place a sample in a cartridge, place the cartridge in the disk-based cartridge reading system, and actuate a button or other input to instruct the computer platform to direct disk-based cartridge reading system to automatically perform steps 150 through step 180. Likewise, actuation parameters pertaining to timing and speed may be pre-programmed for automatic execution by the disk-based cartridge reading system.

The indication of a diagnosis by the detection or lack of fluorescence may be transmitted by any suitable means. In some embodiments, the indication of a diagnosis may be displayed on a digital readout on the cartridge reading system, or displayed wirelessly on a companion device, such as a smartphone or computer.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art may, using the preceding description and the following illustrative examples, utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Example 1 : Malaria Diagnosis

Malaria is a devastating infectious disease and kills more than half a million human lives annually. Accurate and sensitive diagnosis of malaria is required for timely treatment of the infection and prevention of transmission of the malaria parasites. The proposed molecular diagnostic system represents first-of-its-kind, portable, standalone and smart-phone interfaced molecular diagnostic system. The greatest challenge in point-of-care (PoC) molecular diagnosis largely lies in sample preparation process, which requires various dedicated instruments commonly used in the laboratory settings. The present portable system and disposable microfluidic cartridge disks are a paradigm shift in molecular diagnosis. It simplifies the complex DNA analysis and offers unprecedented low-cost and accurate diagnosis performance in a quick and automatic fashion. Rapid and accurate molecular diagnosis of malaria will not only greatly improve potential outcomes of interventions, but also play a major role in the prevention of disease transmission. With a standalone device of versatile connectivity, the portable molecular diagnostic device puts the power of a full diagnostic laboratory into the palms of healthcare workers' hands. A rapid, sensitive and accurate testing of malaria in the field enables healthcare workers to prescribe the right drugs first time. It allows for delivering accurate diagnostic testing to remote, underserved and resource-limited communities. The superior sensitivity and specificity of the molecular diagnosis compared with the conventional microscopy-based diagnosis allow for the identification and treatment of submicroscopic parasite carriers, which are important sources of malaria transmission, and thus will prevent further spreading of the disease.

Malaria in humans is caused by four main Plasmodium species

(Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale) and the zoonotic parasite Plasmodium knowlesi, which is found in many Southeast Asian countries. Despite extensive international efforts, malaria still causes a staggering 243 million cases annually worldwide with half of the world's population remaining at risk of malaria. Malaria diagnosis - detection of malaria parasites in the peripheral blood of patients is required for proper treatments of the infections. Current malaria diagnosis in endemic field settings relies exclusively on microscopy and RDTs. However, both methods have a detection limit of -50 parasites^L; thus patients presenting with lower parasitemias are often missed. So far, satisfactory performance of RDTs has only been achieved for diagnosing P. falciparum malaria. Furthermore, most RDTs for P. falciparum malaria are based on detection of the histidine-rich protein 2 (HRP2) of the parasite. Hence, the deletion of the hrp-2 gene in some P. falciparum populations resulted in a substantial proportion of the malaria cases misdiagnosed as false negatives.

Molecular methods based on nucleic acid amplification are much more sensitive with detection limits approaching ~1 parasite^L. This level of sensitivity enables healthcare workers to identify asymptomatic malaria carriers of malaria parasites, who may serve as reservoirs for continued transmission. Asymptomatic infections often persist at very low parasite densities, below the threshold of detection by microscopy or RDTs. The detection of malaria parasites in this sub-patient population enables targeted control and is essential for the malaria elimination campaigns currently unfolded in many malaria endemic countries.

Current state-of-the-art nucleic acid amplification is polymerase chain reaction (PCR). In PCR, two specific oligonucleotide primers are used to amplify the target sequence. Repetitive cycles of denaturation, annealing and extension result in doubling in the number of target sequences at the end of each cycle. There is an exponential increase in the amount of target sequence over time, as it proceeds through different cycles of amplifications. Though PCR is a highly sensitive and specific method for identification of infection, its requirement for well-equipped laboratories coupled to a well-established cold chain to preserve reagents and thermal cycling inevitably prevent PCR from being used in rural treatment centers far away from reference laboratories. As a result, molecular methods for malaria diagnosis and elimination are mostly used for epidemiological researches and rarely used in field settings, especially in resource-limited countries. LAMP: a diagnostic tool for malaria elimination

As an isothermal amplification technique, Loop Mediated Isothermal Amplification (LAMP) emerges as a very attractive technology for PoC testing. LAMP uses 4-6 primers recognizing 6 distinct regions of the target DNA. A strand-displacing DNA polymerase (Bst) initiates synthesis and 2 of the primers form loop structures to facilitate subsequent rounds of amplification. The major advantage associated with LAMP technique is that nucleic acid amplification can be carried out under isothermal conditions. It is immune to inhibitors present in samples than PCR without

compromising the sensitivity and specificity. These properties make LAMP adoptable for field level diagnosis.

The first attempt of using LAMP for malaria detection in laboratory settings shows that P. falciparum can be correctly diagnosed with heat-treated blood samples. The shorter reaction time without compromising the sensitivity and specificity, and the independence from the use of thermocycler makes LAMP suitable for diagnosis in low facility laboratory settings. However, recent attempts of using LAMP in field settings still need peripheral instrument (such as heating block, water bath, vortex mixer, micro-centrifuge, turbidimeter or fluorometer) to facilitate the sample preparation and results reading. A truly standalone, field-deployable instrument for molecular diagnosis without any infrastructure is still not available.

The major challenges in realizing a standalone 'sample-in-answer-out' instrument for field molecular testing lie in the following aspects: (1) lack of efficient and reliable sample preparation process; (2) lack of intelligent reagent delivery and reaction scheme, (3) lack of a low power consumption system for thermal management; and (4) lack of objective and user-independent results-interpretation. The present study tackles these challenges by realizing a first-of-its-kind, portable, standalone, and smart-phone interfaced molecular diagnostic system.

LAMP detection of P. falciparum species

Initial loop mediated isothermal amplification (LAMP) assay using lab- extracted DNA samples from three standard isolates (strain: Pf529, 3D7, and D10) of Plasmodium falciparum species were validated. Using in-house designed primers targeting the 18S rRNA genes of P. falciparum, the LAMP assay was successfully performed and sensitivity was verified towards Plasmodium falciparum species. An in- house LAMP protocol was developed using calcein as indictor dye. A typical LAMP assay was carried out in a 25^L reaction mixture containing 1 μL of the target DNA, 20 mM Tris-HCl, 10 mM (NH₄)₂S04, 50 mM KC1, 8 mM MgSC , 0.1% Tween 20, 0.2 uM each of F3 and B3 primer, 0.8 μΜ each of LF and LB primer, 1.6 μΜ of FIP and BIP primer, 1.4 mM of each dNTP, 8 unit of the large fragment of Bst DNA polymerase, 25 μΜ of Calcein, and 750 μΜ of MnCh. Figure 8 shows the LAMP assay results under the illumination of a blue LED (wavelength 480 nm), in which a clear bright fluorescence indicates a positive reaction.

Portable molecular diagnostic system design

Arduino microcontroller based components, including the mechanical components, temperature control components, optical components, and connectivity components (display, GPS and Bluetooth), have been prototyped as depicted in Figure 4 A through Figure 4C.

A custom-built printed circuit board (PCB) integrates all the subcomponents involving controlling, commanding, and connectivity into a single small form factor board. The geometrical size of the PCB board is ~ 2 inch x 1 inch. The power consumption is designed to be less than 10 Watt so that a 20000 mAh lithium battery can continuously run the portable system for >10 hours with 5 volts as the driving voltage.

A self-containing package for housing the optical system and the mechanical system is manufactured with black low weight material and machined using laser cutters. In one iteration, the geometrical size and weight of the integrated box is 8 " x 4 " 4 " and ~ 2 lb s, re specti vely .

Open-source Arduino Software (IDE) is used to develop the control software running the microcontroller. The design integrates all control modules that integrates the DNA extraction, purification, amplification, detection, and results interpretation into a single workflow.

Cartidge design

Test cartridges are designed and loaded with all required reagent in a sealed manner (Figure 5). Each cartridge disk has several different chambers and can accommodate several tests. The pre-loaded reagents are sealed from each other by capillary valves and hydrophobic barriers in microfluidic channels. pH switchable magnetic beads are used as the solid-phase extraction platform. The actuation of these magnetic beads in the cartridge disk is driven by a servo-motor, a similar fashion that is used in a CD player.

A plastic lamination technique is used to fabricate the reagent cartridge disk. Top, space and bottom layers are patterned in sterilized material through the use of a laser cutter. These three layers are laminated together by using pressure sensitive acrylic tapes (3M) after loading of the corresponding reagents.

Before laminating and sealing the disposable cartridge, a fixed volume of reagents (-25-100 pL) is loaded into separate chambers of the cartridge by manual pipetting. The reagent includes lysis buffer, magnetic bead binding buffer, washing buffer, and the LAMP reaction mix. The reagents can be preloaded in the laboratory or done in the field.

Validation of the performance of the reagent-loaded cartridge disk will determine: reagent long-term storage, the temperature and humanity effect on the reagent storage, the stability under mechanical shaking and during transport, and the shelf time under proper storage.

Example 2: Field-deployable mobile molecular diagnostic system for malaria at the point of need

Malaria, a mosquito-borne parasitic disease, has been one of the oldest fatal infectious diseases in human history (Enayati, A. et al., Annual Review of

Entomology, 2010, 55:569-591; Greenwood, B. et al., Nature, 2002, 415:670-672; WHO, World Malaria Report 2015, 2015, 1-255; Bhatt, S. et al., Nature, 2015, 526:207-211). Despite extensive malaria control efforts, it still infects -250 million people in the world per year with the remaining half of the world's population at risk (WHO, World Malaria Report 2015, 2015, 1-255; Bhatt, S. et al., Nature, 2015, 526:207-211). For malaria control, rapid, accurate and highly sensitive diagnosis is essential for delivering effective chemotherapies. Currently, malaria diagnosis under field settings relies exclusively on traditional microscopy and rapid diagnostic tests (RDTs) with a detection limit of 50 - 100 parasites/[iL (Vallejo, A.F. et al., Plos Neglected Tropical Diseases, 2015, 9:e3453; Modak, S.S. et al., Infect Dis (Auckl), 2016, 9: 1-9; Wongsrichanalai, C. et al., American Journal of Tropical Medicine and Hygiene, 2007, 77: 119-127; Hopkins, H. et al., Journal of Infectious Diseases, 2013, 208:645-652). Such a detection limit would inevitably miss malaria cases with much lower parasitemias, which are especially common in

asymptomatic parasite carriers (Aydin-Schmidt, B. et al., Plos One, 2014, 9:el03905; Leslie, T. et al., British Medical Journal, 2012, 345:e4389; Pirnstill, C.W. et al., Scientific Reports, 2015, 5: 13368; Wu, L. et al., Nature, 2015, 528:S86-S93). Thus, malaria diagnostic tools with significantly improved sensitivity are urgently needed for endemic settings, especially for regions planning for malaria elimination.

Modern nucleic acid testing (NAT) methods of malaria detection enable much higher sensitivity with a detection limit of approximately 1 parasite^L (Hopkins, H. et al., Journal of Infectious Diseases, 2013, 208:645-652; Wu, L. et al., Nature, 2015, 528:S86-S93), which is highly desirable for identifying asymptomatic infections (Vallejo, A.F. et al., Plos Neglected Tropical Diseases, 2015, 9:e3453; Modak, S.S. et al., Infect Dis (Auckl), 2016, 9: 1-9; Aydin-Schmidt, B. et al., Plos One, 2014, 9:el03905; Wu, L. et al., Nature, 2015, 528:S86-S93; Morris, U. et al., Malaria Journal, 2015, 14: 1-6).

Sensitive detections of malaria parasites in these subpopulations, which are considered as important reservoirs of transmission, are particularly important for malaria elimination (Greenwood, B. et al., Nature, 2002, 415:670-672; Wu, L. et al., Nature, 2015, 528:S86- S93). Among various molecular amplification assays, loop-mediated isothermal DNA amplification (LAMP) has emerged as a promising technology for field use due to its simplicity, rapidness, sensitivity and specificity (Vallejo, A.F. et al., Plos Neglected Tropical Diseases, 2015, 9:e3453; Hopkins, H. et al., Journal of Infectious Diseases, 2013, 208:645-652; Aydin-Schmidt, B. et al., Plos One, 2014, 9:el03905; Han, E T. et al., Journal of Clinical Microbiology, 2007, 45:2521-2528; Polley, S.D. et al., Journal of Clinical Microbiology, 2010, 48:2866-2871; Safavieh, M. et al., Acs Biomaterials- Science & Engineering, 2016, 2:278-294). Major advantages of using LAMP include its high specificity, robustness against inhibitors, and fast amplification (Tomita, N. et al., Nature Protocols, 2008, 3 :877-882; Goto, M. et al., Biotechniques, 2009, 46: 167-172; Notomi, T. et al., Journal of Microbiology, 2015, 53 : 1-5). Unfortunately, most LAMP - based diagnosis still involves bulky and costly peripheral equipment, and skilled technicians are often required for manually operating the instrument (Hopkins, H. et al., Journal of Infectious Diseases, 2013, 208:645-652) and performing multiple steps of sample preparation (Kim, J. et al., Integrative Biology, 2009, 1 :574-586; Myers, F.B. et al., Plos One, 2013, 8:e70266; Liao, S.C. et al., Sensors and Actuators B-Chemical, 2016, 229:232-238; Liu, C.C. et al., Plos One, 2012, 7:e42222). Moreover, basic

infrastructures such as electricity for powering instruments are often limited in remote malaria clinical settings (Choi, S., Biotechnology Advances, 2016, 34:321-330; Abel, G., Expert Review of Molecular Diagnostics, 2015, 15:853-855; Jung, W.E. et al.,

Microelectronic Engineering, 2015, 132:46-57). Therefore, there is a strong desire to develop a molecular diagnostic system that can be more easily deployed to remote malaria endemic areas. Although extensive efforts have been undertaken towards this goal (Yang, K. et al., Lab on a Chip, 2016, 16:943-958; Hu, J. et al., Biotechnology Advances, 2016, 34:305-320), a true "sample-in-answer-out" NAT system with real-time quantitative capability has yet to be developed.

A field-deployable molecular malaria diagnostic platform should possess the following attributes: i) standalone and portable for field applications; ii) true sample- in-answer-out without much user intervention; iii) seamlessly integrated and automated DNA sample preparation, iv) real-time quantitative fluorescence detection; v) rapid and suitable for diagnosis in clinical settings; and vi) much higher sensitivity allowing detection at low parasitemias. The following study reports the design of a molecular diagnostic system for malaria, which consists of a small-footprint analyzer and disposable microfluidic compact discs that are preloaded with molecular reagents for the LAMP assay. With minimal manual work, the system delivers sensitive molecular diagnostic results directly from a small volume of blood samples within 40 minutes without any requirement of laboratory infrastructures. The standalone and user-friendly instrument is highly promising for sensitive malaria diagnosis in field settings. The materials and methods are now described. Culture of malaria-infected blood

P. falciparum 3D7 was maintained in complete RPMI 1640 medium with type 0+ human red blood cells (RBCs) as described previously (Trager, W. et al., Science, 1976, 193 :673-675; Ponnudurai, T. et al., Trans R Soc Trop Med Hyg, 1982, 76:242-250). The parasite culture was synchronized by treatment of ring-stage parasites with 5% D-sorbitol (Lambros, C. et al., J Parasitol, 1979, 65:418-420). The fresh complete medium was replaced on a daily basis and parasitemia was assessed by Giemsa- stained blood smears. In order to mimic the whole blood sample obtained from patients, the parasite culture at the ring stage of various parasitemia was adjusted to around 45% hematocrit for analysis.

Instrumentation

The assembled and exploded views of the system are shown in Figure 4A and Figure 4B. The instrument was designed in SolidWorks and can be quickly prototyped in-house with a 3D printer. The whole system is of a small footprint

(12x13x13 cm). It is powered by a rechargeable lithium-ion battery allowing for 14 hours of continuous operation before recharging. The functional modules inside the system are illustrated in the block diagram (Figure 6).

Real-time optical subsystem - on the excitation side, the LED light source

(λ= 488 nm, C503B-BCN-CV0Z0461, Newark) was guided towards the reaction chamber through a polymer optical fiber (#02-538, Edmund). The optical fiber and LED were self-aligned by a customized adapter to achieve optimal light coupling efficiency. On the detection side, the emission light from the reaction chamber was coupled to the optical sensor (TCS34725, Digi-Key) by the optical fiber. The incidence of excitation

LED light is perpendicular to the optical sensor to minimize the diffracted excitation light into the optical sensor and thus increasing the signal-to-noise ratio.

Thermal subsystem - the thermal module consists of an aluminum heating plate, a Peltier heater, and a thermocouple. The Peltier heater was attached to the bottom side of the aluminum heating plate by thermal adhesive to minimize a temperature gradient. A microprocessor controlled feedback system was used to maintain a desired constant temperature (65 °C). For accurate temperature reading, a mini-thermocouple was embedded inside the heating plate. To evaluate the temperature fluctuation, the temperature was monitored for 60 minutes by an external independent thermocouple module (NI-9211, National Instruments). Figure 9 shows the temperature on the aluminum heating plate can reach the set temperature (65 °C) within 40 seconds and continuously maintain a temperature between 64.5 °C to 66.5°C. Desired reaction temperature was maintained by the feedback control during the DNA amplification process.

Mechanical and interfacing subsystems - an embedded microcontroller unit (MCU) operates the whole system to perform all required isothermal assay steps including automated sample preparation, nucleic acid amplification, and real-time detection. An LCD provides a user-friendly interface for instrument status and data display. In addition, a low power Bluetooth module was incorporated for easy data connectivity.

Tube validation of nucleic acid preparation with magnetic bead-based method

A reference experiment was performed to confirm compatibility of ChargeSwitch forensic DNA extraction/purification kit. To validate the magnetic bead- based DNA extraction/purification method, tube-level sample preparation was manually carried out by pipetting (Figure 10A). In step 1, 20 μΙ_, of sample (blood) was initially dispensed to the tube, which contains 1 mL of lysis buffer and 10 μΙ_, of Proteinase K. This mixture was incubated at room temperature for 2 minutes to lyse the malaria parasites and RBCs. In step 2, 200 μΙ_, of purification buffer and 20 μΙ_, of magnetic beads were introduced by pipetting. In this step, the negatively charged target DNA bind to the positively charged magnetic beads (pH 5.0). In step 3, the DNA-carrying magnetic beads were enriched by a permanent magnet and the remaining supernatant was removed. Then 500 μΙ_, of washing buffer was introduced to remove possible inhibitors. In step 4, 150 μΙ_, of elution buffer was used to unbind the DNA from the magnetic beads due to charge repulsion (pH 8.8). In step 5, 1 μΐ. of purified DNA was introduced to the LAMP master mix, which was transferred to the reagent compact disc and was run on the instrument for real-time amplification. All amplification curves of infected RBC samples showed clear exponential DNA amplification between 25 to 35 minutes (Figure 10B). This result confirms the success of the magnetic bead-based method for malaria DNA extraction and purification.

Microfluidic reagent compact disc design and fabrication

The microfluidic compact disc consists of top, spacer, and bottom poly(methyl methacrylate) (PMMA) layers laminated with adhesive solvent (Figure 3 A). Each layer was designed in AutoCAD (diameter of 9.6 cm) and patterned by a CO2 laser cutter (Epilog Helix 24 Laser System) with a power of 100%, a speed of 30% (for the top/bottom of 0.8 mm thick) and 60% (for the spacer of 0.8 mm thick), and a frequency of 5000 Hz. The patterned top, spacer, and bottom polymethyl methacrylate (PMMA) layers were initially washed with detergent to remove residues from laser cutting, then laminated with adhesive solvent. The assembled disc was cleaned twice with 2% sodium hypochlorite (NaOCl) and distilled water respectively to eliminate inhibitory substances, which could cause chemical interference. Each assembled disc accommodates three independent testing units. Each unit consists of five chambers: a DNA binding chamber (with an inlet for sample input), a washing chamber, a reaction chamber, and two valving chambers (Figure 3B). The valving chambers were filled with FC-40 oil or air. The FC- 40 oil, which seals the reaction chamber, helped prevent master mix evaporation during the thermal process. The air-filled valve was surface treated with water-oil repellent to create a barrier for the amphiphilic lysis buffer. The inlets for sample input were sealed by pressure-sensitive adhesive (PSA) tapes.

Integrated nucleic acid sample preparation on the compact disc

A commercially available DNA purification kit (Invitrogen ChargeSwitch^® forensic DNA purification kit) was used for isolating P. falciparum DNA from whole blood lysates. The lysis buffer, binding buffer, and washing buffer were used as received. The 10 μL· of human whole blood samples spiked with cultured P.

falciparum were collected into a 1.5 mL microcentrifuge tube containing 1000 iL of lysis buffer and 10 μΐ. proteinase K. After incubating at room temperature for 2 min, 180 μΐ. lysates were introduced into the binding chamber through the inlet hole. There are three independent testing units on the compact disc. Each testing unit on the compact disc consists of (1) 30 μΐ. binding buffer and 10 μΐ. magnetic beads in the binding chamber, (2) 150 μΐ. washing buffer in the washing chamber, and (3) 25 iL LAMP master mix in the reaction chamber (Figure 3B). Since the surface charge polarity of the magnetic beads is pH dependent and the surface charge polarity of DNA is negative for a wide range of pH values (Lien, K.Y. et al., Microfluidics and Nanofluidics, 2009, 6:539- 555), the magnetic beads can attract or repel the nucleic acids by the different pH values of the preloaded buffer solution (Figure 3C). The magnetic beads were actuated by rotating the compact disc against a stationary magnet. In a control sequence, the DNA- carrying beads were directed into the different chambers until the target DNAs were eluted in the reaction chamber (Figure 3C). The whole process automatically prepared high-quality DNA samples from the human whole blood in less than 10 minutes.

Loop-mediated isothermal DNA amplification

The LAMP reaction mix consists of isothermal buffer (20 mM Tris-HCl, 10 mM (NH₄)₂S04, 50 mM KC1, 2 mM MgSC , 0.1% Tween 20, pH 8.8), P. falciparum- specific primer set (5 pmol F3 and B3, 40 pmol FIP and BIP, 20 pmol LF and LB, Figure 1 IB), MgS0₄, calcein, MnCl₂, deoxyribonucleotide triphosphates (dNTPs), Est 2.0 DNA polymerase, DNA template, and PCR grade H₂0 (Figure 11 A. The LAMP assay was performed at a constant temperature (65°C maintained by the analyzer) (Figure 9). Six target-specific primers targeting mitochondrial gene were synthesized (Integrated DNA Technologies) to specifically amplify the 213-bp region of the P. falciparum DNA (Figure 12, Figure 11B) (Polley, S.D. et al., Journal of Clinical Microbiology, 2010, 48:2866-2871). Data analysis During the amplification process, the fluorescence readings were acquired every 2.5 seconds. The first 5 min of the signal was averaged to obtain the background noise level, which was then subtracted from the raw fluorescence readings to form a processed signal. The processed signal was further smoothed by averaging a fixed number of consecutive data points. A threshold time (Tt) was defined at the time when the slope of measured RFU (dRFU/dt) reached the peak (Figure 13 A, Figure 13B). The threshold RFU value was experimentally determined at 400 for positive/negative decision.

The results are now described. System operation

Figure 14 shows the operation of the system from the whole blood sample to the molecular diagnostic results. The nucleic acid testing procedure includes the following seamlessly integrated steps: (1) collecting malaria-infected blood into 1.5 mL microcentrifuge tube containing 1000 μΐ. of lysis buffer and 10 μΐ. proteinase K, mixing the content thoroughly, and incubating at room temperature for 2 minutes; (2)

transferring 180 μΐ. of the lysate into the compact disc through a loading inlet which was sealed after loading; and (3) inserting the compact disc into the instrument. The instrument automatically performs all steps including DNA purification, elution, amplification, and real-time detection. The diagnostic result is shown on an LCD screen or (optionally) on a smartphone through Bluetooth connectivity. The turnaround time from sample to answer is less than 40 minutes. Although the lysis process could also be incorporated into the compact disc, an off-chip lysis was adopted to ensure the system' s compatibility with other sample types (e.g., swab samples).

Passive valve on the microfluidic compact disc

One major challenge in applying microfluidics towards point-of-care testing is the need for peripheral tubing and pumping systems to drive the liquid movement (Martinez, A.W. et al., Analytical Chemistry, 2010, 82:3-10; Kong, L.X. et al., Microfluidics and Nanofluidics, 2015, 18: 1031-1037). One exception is the centrifugal type microfluidic platform, where the reagent can be preloaded and driven by centrifugal forces (Madou, M. et al., Annual Review of Biomedical Engineering, 2006, 8:601-628). The preloaded reagents rely on passive valves to prevent mixing (Park, J.M. et al., Lab on a Chip, 2007, 7:557-564; Gorkin, R. et al., Lab on a Chip, 2010, 10: 1758-1773).

However, fluid control and separation in the centrifugal platform were challenging because an identical centrifugal force field is applied to all of the liquids on the disc within which a different flow rate is needed (Kong, L.X. et al., Microfluidics and

Nanofluidics, 2015, 18: 1031-1037; Burger, R. et al., Microfluidics and Nanofluidics, 2012, 13 :675-681). In addition, centrifugal force is non-linear in nature and needs rotational frequencies in the range of several thousand revolutions per minute (RPM) (Madou, M. et al., Annual Review of Biomedical Engineering, 2006, 8:601-628). The electrical power needed to drive this motion is tremendous.

The microfluidic reagent disc used in the system does not rely on the centrifugal force to move the liquid. Instead, the DNA-carrying magnetic beads are actuated against the stationary reagent droplets. The reagents were preloaded and separated on the microfluidic reagent disc by teeth-shaped passive valves (Figure 3C). Structural pinning effect (Gao, L.C. et al., Langmuir, 2006, 22:6234-6237; Oner, D. et al., Langmuir, 2000, 16:7777-7782) and modified surface tension are the underlying principles that enable the teeth-shaped valves to securely hold the liquid in each chamber. The pinning effect refers to a fact that a sharp bending angle (a) of the teeth structure radically increases the liquid/vapor interface area and raises the activation energy, which prevents fluid to overcome the barrier (Figure 15 A) (Gao, L.C. et al., Langmuir, 2006, 22:6234-6237). The enhanced surface tension is another important aspect of our passive valve structure. The valve surface was treated with water-oil repellent to increase the activation barrier by introducing a higher surface tension (Gao, L.C. et al., Langmuir, 2006, 22:6234-6237), which also helps circumvent cross-contamination during sample preparation.

To demonstrate the robustness of the passive valve for preventing the reagents from mixing under the harsh mechanical vibration, a drop test was performed on the microfluidic compact disc. Three different colors of food dyes were preloaded into each reagent chamber for visualization of any liquid movement. Each reagent-loading hole was sealed with pressure-sensitive adhesive (PSA) to prevent leakage. The disc was dropped from a height of 20 cm along a guiding rod towards a rigid surface for 25 times. The disc was inspected every five drops with naked eyes to confirm functionality of the valve. The result showed that the teeth-shaped valve endured 25 consecutive drops without reagents mixing (Figure 15B). In addition, the robustness of the passive valve was also validated through hand agitation to the microfluidic disc. Assay validation on the compact disc

To validate the designed LAMP assay and the module level function (fluidic, thermal, mechanical, optical) of the instrument, the LAMP assay in the microfluidic compact disc on the system was compared side-by-side with a real-time PCR using purified P. falciparum genomic DNA sample. To this end, purified P.

falciparum genomic DNA (gDNA) was 10-fold serially diluted with Tris-EDTA buffer. LAMP master mix without P. falciparum gDNA was used as a no template control (NTC). As shown in Figure 16 A, the assay was firstly validated by a benchtop real-time PCR machine. A sharp increase in RFU values was observed from three diluted DNA samples while the negative control showed no increase of RFU values (Figure 16A). The real-time performance of the system on the same sample sets is shown in Figure 16B, which clearly demonstrated a distinguishable fluorescence threshold between positive and negative samples within 30 minutes. In addition, the result of the system was quantitative. An inversely proportional relationship between the threshold time and the DNA concentrations (R² = 0.9998) was observed (Figure 16B inset).

To further validate that the amplicons were indeed from the specifically designed targets, a gel electrophoresis analysis of the amplicons was performed in 2% agarose gel. As shown in Figure 16C, the LAMP amplicons showed a clear ladder-like pattern with multiple bands of different molecular sizes due to its inverted-repeat structures (Notomi, T. et al., Nucleic Acids Research, 2000, 28:e63-e63). More specifically, the length of the observed bands corresponds to the integral times of the target sequence (213 bp), indicating the amplified products were specific for the target sequence. In addition, strong green emission (implying positive reactions) can be easily recognized by the naked eyes in the PCR tube under blue LED illumination in the dark environment (Figure 16D). From these results, the LAMP assay was successfully verified against P. falciparum genomic DNA and the modular function of the system.

Integrated sample preparation on the compact disc

High-quality nucleic acid sample preparation is the bottleneck for most "sample-in-answer-out" molecular analysis (Foudeh, A.M. et al., Lab on a Chip, 2012, 12:3249-3266). Before the sample was prepared on the reagent compact disc, the pH- switchable magnetic beads-based method for DNA isolation was validated first in test tubes from the blood sample using the same reagents as on the reagent compact disc. Successful amplification of the tube-extracted DNA samples confirmed the effectiveness of the magnetic bead-based method (Figure 10B). Afterward, sample preparation in the reagent compact disc and the compatibility of the purified DNA with the subsequent LAMP assay were evaluated. To test the DNA extraction efficiency on the reagent compact disc, P. falciparum infected blood samples of different parasitemia (0.2%, 0.02%, and 0.002%), prepared by dilution with healthy blood at 45% hematocrit) were examined. The infected blood samples were directly lysed in the collection tubes before loading into the reagent compact disc (Figure 3B). The following DNA binding, purification, elution, and the amplification were automatically carried out by the system (Figure 3C). As shown in Figure 17, the real-time amplification data for various parasitemia showed a clear quantitative trend, as compared to the lack of amplification signals in the negative control. These results fully verified that the system could work in a "sample-in-answer-out" fashion by seamlessly integrating high-quality DNA

preparation and real-time amplification on a single reagent compact disc.

Assay sensitivity

To evaluate the diagnostic sensitivity (Saah A.J. et al., Annals of Internal Medicine, 1997, 126:91-94) of the system for analyzing the whole blood samples, various parasitemias of P. falciparum-mfected blood (from 2% to 0.00002%, and 2% parasitemia corresponds to -60,000 parasites^L) were prepared by diluting with fresh human RBCs at 45% hematocrit. In addition, the master mix (Figure 11 A) without target DNAs as well as the healthy RBCs (hBRC) were used as negative controls. To evaluate the test-to- test variations, each whole blood sample was examined independently three times. As shown in Figure 18, the amplification curves of the infected RBC samples show clear exponential increases of fluorescence, while those of the negative controls (master mix and hRBC) show no amplification. The amplification threshold time was inversely proportional to the parasitemia (inset of Figure 18), verifying that the system was a quantitative "sample-in-answer-out" system. Parasite DNA in the blood sample at

0.00002%) parasitemia could be successfully detected and quantified, which corresponded to a detection limit of -0.6 parasites^L. Since only 180 μΙ_, out of a total of 1020 μΙ_, lysate was used as the input to the reagent compact disc (Figure 3B), a lower detection limit (-0.1 parasites/ μί) could be achieved if all lysate volume were used (requiring a disc redesign). The detection limit of -0.6 parasites^L of the system is comparable to the benchtop real-time PCR test (-0.7 parasites^L; Perandin, F. et al., Journal of Clinical Microbiology, 2004, 42: 1214-1219) and to the benchtop LAMP test (-2 parasites^L; Ay din- Schmidt, B. et al., Plos One, 2014, 9:el03905). This level of sensitivity is necessary for detecting the early-stage asymptomatic parasite carriers of low parasite densities (Aydin-Schmidt, B. et al., Plos One, 2014, 9:el03905; Slater, H.C. et al.,

Nature, 2015, 528:S94-S101), which are often missed by either the immunoassay based rapid diagnostic tests (RDTs, -100 parasites/ μί; Wongsrichanalai, C. et al., American Journal of Tropical Medicine and Hygiene, 2007, 77: 119-127) or microscopy (-30-50 parasites/ μί; Wongsrichanalai, C. et al., American Journal of Tropical Medicine and Hygiene, 2007, 77: 119-127; Pirnstill, C.W. et al., Scientific Reports, 2015, 5: 13368). Thus, the system is able to deliver ultrasensitive and quantitative molecular answers for malaria infections in remote settings without supporting infrastructures.

The greatest advantage of the molecular test is their ability to detect extremely low-level malaria infections, which are often challenging for microscopy and RDTs. Nevertheless, the greatest hurdle for deploying a molecular test in resource- limited areas is its relatively high cost and the infrastructure investment. The system aimed to address this issue by delivering a sensitive malaria molecular test in a cost- effective way. The prototype instrument presented in this work could be built for a total amount of ~$176 (see Figure 19 for cost breakdown). The disposable reagent compact disc (including the sample preparation and the amplification reagents) costs ~$1.14 per each test (Figure 20). The low cost of this sensitive molecular test provides a great opportunity for the field applications of this mobile molecular diagnostic system.

Moreover, the manufacturing of the instrument is highly scalable in a cost-effective way. The system could be easily reproduced within a day. The instrument-to-instrument variation was small enough to deliver the same quality of quantitative molecular analysis (Figure 21). As a result, the system enables cost-effective malaria molecular diagnosis in resource-limited regions by decreasing the cost, increasing the ease of use, and maintaining high sensitivity. The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:

1. A cartridge device for point-of-care molecular diagnoses comprising:

a substantially planar casing;

at least one chamber within the casing;

at least one microchannel connecting the at least one chamber within the casing; and

at least one inlet in the casing providing access to the at least one chamber;

wherein the at least one chamber and at least one microchannel are aligned in an arc.

2. The device of claim 1, wherein the at least one chamber includes a lysing chamber, a binding chamber, a washing chamber, and a reaction chamber.

3. The device of claim 2, wherein the lysing chamber is preloaded with a volume of lysis buffer.

4. The device of claim 2, wherein the binding chamber is preloaded with a volume of binding buffer with pH-switchable magnetic beads, wherein the binding buffer comprises a pH below 6.5.

5. The device of claim 2, wherein the washing chamber is preloaded with a volume of washing buffer.

6. The device of claim 2, wherein the reaction chamber is preloaded with a volume of LAMP reaction mix, wherein the LAMP reaction mix comprises a pH above 8.8.

7. The device of claim 6, wherein the LAMP reaction mix further comprises a fluorescent dye.

8 The device of claim 1, wherein the at least one microchannel comprises a capillary valve for separating the contents of the at least one chamber.

9. The device of claim 1, wherein the at least one microchannel comprises a hydrophobic coating for separating the contents of the at least one chamber.

10. The device of claim 1, wherein the arc comprises a geometry proportional to a segment of the circumference of a disk in a disk-based cartridge reading system.

11. The device of claim 1, wherein the device is disposable.

12. A disk-based cartridge reading system, comprising:

a disc comprising at least one cartridge slot;

at least one substantially planar cartridge comprising at least one chamber and at least one microchannel connecting the at least one chamber, wherein the at least one chamber and at least one microchannel are aligned in an arc;

a servomotor;

at least one magnet;

at least one heating element;

at least one photodetector; and

at least one light source;

wherein the disk is capable of holding at least one cartridge, and wherein the servomotor is capable of actuating the disk to position the chambers of the cartridge near the at least one magnet, the at least one heating element, the at least photodetector, and the at least one light source.

13. The system of claim 12, wherein the at least one magnet is an electromagnet.

14. The system of claim 12, wherein the at least one light source is one of a laser or an LED.

15. The system of claim 12, further comprising a component selected from the group consisting of: a GPS component, a digital display component, a portable battery power component, a wireless transmitting and receiving component, a

microcontroller component, and a data reading and writing component.

16. A method of point-of-care molecular diagnosis, comprising the steps of:

acquiring a sample;

acquiring a cartridge comprising a lysing chamber with lysing buffer, a binding chamber with binding buffer and magnetic beads, a washing chamber with wash buffer, and a reaction chamber with LAMP reaction mix, wherein the chambers are connected in sequence by a plurality of microchannels;

placing the sample in the lysing chamber with lysing buffer to release sample nucleic acids;

placing the cartridge in a disk-based cartridge reading system comprising a disk, at least one magnet, at least one heating element, at least one photodetector, and at least one light source;

activating the at least one magnet and actuating the disk such that the magnetic beads and binding buffer are drawn into the lysing chamber by the at least one magnet, wherein the magnetic beads bind the sample nucleic acids;

actuating the disk such that the magnetic beads and sample nucleic acids are drawn into the buffer chamber, the washing chamber, and the reaction chamber by the at least one magnet;

deactivating the magnet and actuating the disk to position the reaction chamber near the at least one heating element to carry out a LAMP reaction; and actuating the disk to position the reaction chamber near the at least one photodetector and the at least one light source to detect fluorescence, wherein the detection of fluorescence indicates a positive diagnosis.

17. The method of claim 16, wherein an amount of 10 to 100 μΙ_, of sample is acquired.

18. The method of claim 16, wherein the sample is pre-lysed prior to placement in the lysing chamber of the cartridge.

19. The method of claim 16, wherein the actuation timing and actuation speed of the disk are adjusted based on the type of diagnosis being performed.

20. The method of claim 16, wherein the heating element maintains a temperature of 60° C for the duration of the LAMP reaction.