Description:FIELD OF THE INVENTION
[0001] The present invention relates to the field of soil microbiology, and more particularly, the present invention relates to the water testing system for pathogenic microbes.
BACKGROUND FOR THE INVENTION:
[0002] The following discussion of the background to the invention is intended to facilitate an understanding of the present invention. However, it should be appreciated that the discussion is not an acknowledgment or admission that any of the material referred to was published, known, or part of the common general knowledge in any jurisdiction as of the priority date of the application. The details provided herein the background if belongs to any publication is taken only as a reference for describing the problems, in general terminologies or principles or both of science and technology in the associated prior art.
[0003] Safe and clean drinking water is among the most significant global health concerns, especially in emergency relief situations such as natural disasters or floods or in areas of low resource, remote communities, etc. Aerobic odorless and colorless microbial contamination of water by such agents as Vibrio cholerae, Cryptosporidium, Escherichia coli, and Giardia lamblia and the resulting disease from cholera diarrhea to acute cholera and other potentially fatal diseases are some of the most intransigent of these issues. While most governments and agencies are attempting to screen and monitor the water, detection is hindered by several factors that slow and minimize its effectiveness.
[0004] The current water testing procedure typically entails drawing and sending water samples to the best labs. They are generally culture-based, so long as three days must pass before findings are obtained. In the meantime, dirty water can be held after being used, spreading disease on a massive scale. In addition, the procedures involve trained technicians, strict sample-handling techniques, and lab access to expensive and sensitive equipment. They are unavailable in disaster or remote locations, and rapid and regular testing is impossible. No matter how widely newer and more sophisticated molecular methods such as PCR (polymerase chain reaction) are being used, these are based on high-tech machinery and aseptic laboratory facilities. They are rigid, energy-consuming, and involve complex processes beyond the capabilities of non-professionals. Thus, their application in the region where quicker decision-making is most valuable is restricted. Moreover, most of the rapid tests available today, e.g., lateral flow devices, can detect only a single type of pathogen and are not sensitive to detect low levels of contamination. They are also prone to provide a yes/no response and can be susceptible to false positives or false negatives. They also typically cannot talk back to computerized systems to provide results, monitor trends, or issue timely public health alerts. In this case, having an instant field-deployable system for water analysis addresses several key problems.
[0005] In light of the foregoing, there is a need for a water testing system for pathogenic microbes that overcomes problems prevalent in the prior art. By integrating immunological detection (which detects the presence of a pathogen by looking for surface markers) and nucleic acid detection (which tests for the DNA or RNA of the pathogen), this technology enhances test sensitivity and specificity. This technology uses electricity, a pipette, and a laboratory setup-free paper platform, perfect for field deployment immediately.
OBJECTS OF THE INVENTION:
[0006] Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows.
[0007] The principal object of the present invention is to overcome the disadvantages of the prior art by providing the Water testing system for pathogenic microbes.
[0008] Another object of the present invention is to provide a water testing system for pathogenic microbes that combines some very potent concepts into a single, portable, and user-friendly device that enables any individual, whether a trained scientist or a villager from some far-flung village, to detect water for dangerous germs at speed and with accuracy.
[0009] Another object of the present invention is to provide a water testing system for pathogenic microbes that combines two forms of germ detection tests in one test: one for surface markers (immunoassay) and another for genetic material (DNA or RNA). By combining both techniques into a single strip, this invention makes the test far more sensitive and precise.
[0010] Another object of the present invention is to provide a water testing system for pathogenic microbes that can detect even trace amounts of germs in water that other tests cannot detect.
[0011] Another object of the present invention is to provide a water testing system for pathogenic microbes, wherein when the water sample is deposited, it gets directed automatically down the strip—no buttons, no wires—triggering each stage in the sequence.
[0012] Another object of the present invention is to provide a water testing system for pathogenic microbes that makes it possible for the test to be given where electricity and equipment are unavailable, only by the naturally generated heat of the test itself.
[0013] Another object of the present invention is to provide a water testing system for pathogenic microbes that has sections that sterilize and destroy any leftover germs or genetic material and, therefore, are less likely to infect another person when used.
[0014] Another object of the present invention is to provide a water testing system for pathogenic microbes that unifies accuracy, user-friendliness, safety, and computer sophistication into one piece of equipment that no other water test has ever been able to accomplish as a whole.
[0015] Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
SUMMARY OF THE INVENTION:
[0016] The present invention provides a water testing system for pathogenic microbes.
[0017] The originality of this invention is how it combines some very potent concepts into a single, portable, and user-friendly device that enables any individual, whether a trained scientist or a villager from some far-flung village, to detect water for dangerous germs at speed and with accuracy. Although there are other machines to check water, none combine many essential qualities in one simple-to-use strip that does not require electricity, lab tools, or professional knowledge. This is not just a new gadget; it is a better, more complete solution to real water safety problems.
[0018] What is particularly original about this invention is that it combines two forms of germ detection tests in one test: one for surface markers (immunoassay) and another for genetic material (DNA or RNA). Most field tests have used one of these forms, failing to identify infections or creating false positives. By combining both techniques into a single strip, this invention makes the test far more sensitive and precise. It can detect even trace amounts of germs in water that other tests cannot detect. The device has a further innovative feature: a paper system with "channels" to direct the water sample automatically through all test stages. Dry chemicals are incorporated within the paper at micro-locations. When the water sample is deposited, it gets directed automatically down the strip—no buttons, no wires—triggering each stage in the sequence. All the testing is smooth and relatively simple for non-scientists and non-clinical individuals.
[0019] Also new is how this test regulates heat. Unlike other tests that need equipment to keep the test at a set temperature, this one uses special materials that start heating automatically as soon as the test begins. This new use of materials makes it possible for the test to be given where electricity and equipment are unavailable, only by the naturally generated heat of the test itself. The most significant improvement is in smartphone compatibility. The test contains a printed reference color scale, such as an integral color ruler, so when someone takes a photo of the strip, it can be readily read using a cell phone application. The app considers parameters like light or camera sensor performance, providing sharp responses and even alerts, e.g., "Safe to Drink" or "Boil Water First." The app also measures time, geolocation, and outcome, which allows public health officials to look at water safety in real-time from numerous geographies. Most importantly, the strip is rendered safe to use following the test. It has sections that sterilize and destroy any leftover germs or genetic material and, therefore, are less likely to infect another person when used. Overall, the innovative aspect of this invention is that it unifies accuracy, user-friendliness, safety, and computer sophistication into one piece of equipment that no other water test has ever been able to accomplish as a whole. It is not infinitely improved; it is one giant leap towards clean water testing accessibility for all locations everywhere.
[0020] This invention is an easy, field-portable water test kit so that individuals can test whether their water is drinkable. It is particularly convenient where clean water is not readily available, or there is no lab and electricity, i.e., in remote villages, disaster situations, or wilderness camps. The aim is to make it so individuals can detect poisonous germs in water without training or equipment. Millions of individuals get sick yearly because they drink unclean water with trace microorganisms such as bacteria and parasites. They cause severe health diseases such as diarrhea, cholera, and other gastroenteritis illnesses. Water bacteria analysis usually takes long days and requires a specific laboratory, trained professionals, and costly equipment. Waiting time is unsafe and can cause individuals to lose their health, particularly in cases of urgency. This invention is much quicker and easier. It uses a very thin sheet of paper containing special chemicals that trap and identify germs. One adds a water sample to the paper, flowing through narrow channels. The channels contain dry chemicals that get wet when water gets into them. Because the strip contains chemicals when the water runs through it, it will naturally cause a few things: trap the germs, do some specialized genetic material testing, and emit a visible trace indicator if they detect something yucky. The result is a color shift or very low-order strip lighting. Creating a simplified reading, an application on a cell phone can read the strip using the phone camera. The device gets the signal and gives a message to the user to inform him or her if he or she should or should not use the water, generally in plain messages like "Boil Before Use" or "Do Not Drink." It even suggests medical action depending on the threat level and location. The biggest plus of the device is that brilliance, speed, and convenience have all been put into one. It provides results within an hour, is portable to any place in the pocket, and is machineless and wireless. It has safety features that kill remaining germs after finishing the test, so there will be no transmission of contamination. The strips may be offered for various germs, and the customers can choose to replace them at intervals with the same base unit; thus, it is cheap. The technology is accessible to families that drink river or well water, emergency responders, tourists, or government officials monitoring real-time water quality. It is a democratizing means of putting control of their water safety into individuals' hands and staying healthy. It is no longer confined to a lab and can instantly and accurately test water, accessible to anyone, anywhere.
BRIEF DESCRIPTION OF DRAWINGS:
[0021] Reference will be made to embodiments of the invention, examples of which may be illustrated in accompanying figures. These figures are intended to be illustrative, not limiting. Although the invention is generally described in the context of these embodiments, it should be understood that it is not intended to limit the scope of the invention to these particular embodiments.
[0022] Figure 1: Battery-operated handheld reader device.
[0023] Figure 2: A test strip showing different regions/channels on which the water sample flows uniformly in one direction due to the capillary action.
[0024] Figure 3: The handheld device shows the UV light lamp, which is battery-operated.
[0025] Figure 4: A test strip showing different components.
DETAILED DESCRIPTION OF DRAWINGS:
[0026] While the present invention is described herein by way of example using embodiments and illustrative drawings, those skilled in the art will recognize that the invention is not limited to the embodiments of drawing or drawings described and are not intended to represent the scale of the various components. Further, some components that may form a part of the invention may not be illustrated in certain figures, for ease of illustration, and such omissions do not limit the embodiments outlined in any way. It should be understood that the drawings and the detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the scope of the present invention as defined by the appended claim.
[0027] As used throughout this description, the word "may" is used in a permissive sense (i.e. meaning having the potential to), rather than the mandatory sense, (i.e. meaning must). Further, the words "a" or "an" mean "at least one” and the word “plurality” means “one or more” unless otherwise mentioned. Furthermore, the terminology and phraseology used herein are solely used for descriptive purposes and should not be construed as limiting in scope. Language such as "including," "comprising," "having," "containing," or "involving," and variations thereof, is intended to be broad and encompass the subject matter listed thereafter, equivalents, and additional subject matter not recited, and is not intended to exclude other additives, components, integers, or steps. Likewise, the term "comprising" is considered synonymous with the terms "including" or "containing" for applicable legal purposes. Any discussion of documents, acts, materials, devices, articles, and the like are included in the specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention.
[0028] In this disclosure, whenever a composition or an element or a group of elements is preceded with the transitional phrase “comprising”, it is understood that we also contemplate the same composition, element, or group of elements with transitional phrases “consisting of”, “consisting”, “selected from the group of consisting of, “including”, or “is” preceding the recitation of the composition, element or group of elements and vice versa.
[0029] The present invention is described hereinafter by various embodiments with reference to the accompanying drawing, wherein reference numerals used in the accompanying drawing correspond to the like elements throughout the description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art. In the following detailed description, numeric values and ranges are provided for various aspects of the implementations described. These values and ranges are to be treated as examples only and are not intended to limit the scope of the claims. In addition, several materials are identified as suitable for various facets of the implementations. These materials are to be treated as exemplary and are not intended to limit the scope of the invention.
[0030] The present invention provides a water testing system for pathogenic microbes.
[0031] The availability of safe and hygienic drinking water is the oldest and most perpetual among all public health concerns in the world, especially in poor environments, rural, far-flung refugee camps, and areas of hazardous natural disasters like floods, earthquakes, and hurricanes. These areas have no facility, central lab, treatment plant, or functioning facilities. People are thus exposed to unknown sources of polluted water with harmful microbial threats. Microbial pathogens cause diseases like gastrointestinal disease, cholera, and diarrheal death in children, the elderly, and immunocompromised persons. The best way to identify microbial contamination of potable water is by collecting water samples and analyzing them in laboratory facilities using the culture method or molecular techniques like the polymerase chain reaction (PCR). Although they take time, 24 hours to a couple of days before making the results public, they are not gratis. They involve sophisticated equipment, highly trained and experienced laboratory technicians with significant training, and specialized laboratory facilities, especially for this. It is rendered impossible by such conditions to employ tests in most terrains where immediate fast water safety testing is required, e.g., disaster areas, slum camps, and rural health post locations.
[0032] Due to such limitations, scientists and engineers have achieved remarkable new capabilities with new field-portable water analysis technologies. The most promising new technologies being developed are nucleic acid amplification technologies and paper microfluidic devices. These emerging technologies draw on the science of microfluidics, in which liquid flows through channels of infinitesimally narrow cross-sections on paper or otherwise to perform complex biological assays without electricity, trained technical personnel, or pipettes. Low cost and minimalism mean being deployable in massive numbers in the field. Also, nucleic acid amplification techniques like isothermal amplification can be incorporated directly within such paper-based systems to detect extremely trace amounts of pathogen RNA or DNA even without incorporating conventional thermal cycling elements. Some analysis steps are performed within a single, very tiny, closed device with such devices and are extremely time- and complexity-effective for detecting microbes. Others provide mobile phone application support in visually administering tests and real-time reporting of findings into public health surveillance systems.
[0033] The integration of technology, high-end fluid control, inexpensive materials, and high-end biological detection technologies has enabled the placement of low-cost, easy-to-use portable water analysis technology that can potentially transform water safety practices among poor populations. These emerging technologies hold high potential to democratize water testing so that individuals and communities can quickly test their water source and respond rapidly to contamination to avoid disease transmission and loss of life. Among the breakthrough portable diagnostic technologies is the development of an integrated single-use nucleic acid extraction, amplification, and detection paper-based device. This new platform represents a bound and leaps ahead of the simplification of molecular diagnostic processes that are even possible in low-resource settings. The classical processes of nucleic acid-based detection are typically sequential, successive procedures such as sample preparation, nucleic acid extraction, temperature-cycling amplification reactions such as PCR, and detection. All these various steps involve reagents, energy, precise equipment, and trained personnel, none of which are available in field conditions or outstations in most cases. The paper-based integrated device eliminates all such constraints by integrating all the components into a small, user-friendly, self-contained diagnostic device.
[0034] At the heart of the technology lies the ability that this technology holds for helicase-dependent isothermal amplification, a type of DNA amplification in isothermal and not thermal cycling form. It does it better and more inexpensively but at the cost of no longer needing gargantuan. The device is a middleman where nucleic acids are eluted from original samples on the paper strip. Among the requirements for use in the field when cold-chain shipping is too costly or unavailable is why reagents dried within the paper matrix are kept for years at room temperature. The plant uses lateral flow assay (LFA) technology to offer an on-site and immediate visual presentation of test results, like a pregnancy test, that users can self-read without any special training beyond that needed for the general public. Visual markers make user error impossible and enable test results to be readable without special instrumentation. In addition, the test itself is easy; users need to insert a sample and start the reaction in only a few steps, so even those without laboratory science experience can use it.
[0035] The product has also been highly sensitive and specific in detecting microbial pathogens. For example, in validation testing, it would be able to identify Salmonella typhimurium, a prevalent and potentially fatal food- and waterborne disease-causing bacterium, to a 10² CFU/ml level of sensitivity. Notably, the assay performed optimally even in such challenging matrices as wastewater and some foods and was rugged and acceptable for routine application. Also, the whole process, from sample introduction to result interpretation, is accomplished within one hour, and rapid turnaround is essential in public health crises. The frugal diagnostic device intersects frugal innovation, microfluidics, and biotechnology. The device is disposable, low-cost, and handheld; therefore, it is the most desirable device for decentralized testing. Whether in impacted disaster areas, rural health facilities, or through rural village frontline health workers, such organized systems could potentially be able to allow communities to detect and take action upon microbial threats before such had escalated to hazard levels, hence circumventing the escalation of disease transmission, augmenting surveillance, and ultimately safeguarding public health where under threat.
[0036] Another innovation in field-deployable diagnostics is the creation of a paper-based nucleic acid enrichment platform as a field-deployable platform for the rapid and reliable detection of infectious disease-causing pathogens in remote or resource-constrained locations. This platform combines the best molecular biology, microfluidics, and mobile electronics to provide a cost- and energy-efficient diagnosis platform that considerably narrows the sample collection-to-result interpretation time gap. Thus, this platform is an evolutionary leap for off-lab, real-time infectious disease monitoring and control. The device comprises a battery-powered miniaturized all-in-one reader and a paper-based enrichment chip. The enrichment chip recovers nucleic acid from crude biological samples like stool, nasal swabs, or saliva. The chip utilizes proprietary paper matrices that capture the nucleic acid and eliminate the inhibitors and contaminants to produce a highly purified template for downstream amplification. Its simplicity makes the chip light, inexpensive, and simple to mass-produce, a gigantic advantage in the case of public health emergency mass deployment. The sample is inserted into the enrichment chip and read. The user performs the whole nucleic acid amplification process using the isothermal process, like loop-mediated isothermal amplification (LAMP) or recombinase polymerase amplification (RPA). Reactions do not undergo thermocycling as in PCR; therefore, the whole amplification process can be performed at a fixed temperature provided by the reader. Battery power renders the instrument completely grid electricity independent. It can thus be used in off-grid, rural areas or disaster zones where standard laboratory equipment would be impractical or even impossible. This electricity-free handheld test strip is used to quickly detect microbial impurities in drinking water through capillary-based microfluidic technology. This image depicts a low-cost, deployable, accessible water testing system for microbes by passive paper-based microfluidics and smartphone-based digital interpretation. The disposable, easy-to-use, low-cost system makes water testing accessible and allows early detection of pathogens in emergency or resource-poor settings. This point-of-care water quality test strip offers rapid, inexpensive, and reproducible water quality determination without the need for a laboratory. A mobile phone application offers increased reliability, result interpretation, and geotagged data transfer for public health surveillance. Each labelled feature represents a key functional area of the test device. Figure 1 shows the handheld reader device, a battery-operated instrument on which the nitrocellulose strip is placed.
[0037] Figure 1. Battery-operated handheld reader device, on to which the test paper/nitrocellulose strip is placed in the slot [1], grooves for test strip slot [2], which correctly hold the test strip; water sample loading tray slot [3], where the water sample is poured.
[0038] Figure 2. A test strip [4] shows different regions/channels on which the water sample flows uniformly in one direction due to the capillary action.
[0039] The test strip is placed in the slot on the upper surface of the handheld device. It can be placed over the device easily and taken out simply with no trouble.
[0040] A drop or small amount of the water sample (1–3 drops) is placed in the top section. This triggers capillary flow through pre-formed microfluidic channels within the paper. This section provides reliable wicking to downstream detection modules for easy sample delivery. This is done to initiate the diagnostic process. It initiates capillary-driven fluid migration on the paper surface. The material exhibits uniform wicking and does not spill over.
[0041] Figure 3. The handheld device shows the UV light lamp [5], which is battery-operated. When the UV lamp glows, the fluorescent color of the completed test reaction of the test strip can be seen, which cannot be seen by the naked eye. To observe the color change (fluorescent color), a test strip must be kept on the device slot.
[0042] Figure 4. A test strip showing different components, sample loading tray [3], dropper for adding the test sample [6], sample loader and distributor slot [7], channels for equal sample distribution [8], detection lines [9], conjugate pad [10], primary antibodies [11], secondary antibodies [12], dried reagents (labeled nucleic acid probes) [13], test line [14], control line [15], absorbent pad [16].
[0043] As shown in Figure 4, the test water sample is applied to the sample loader and distributor slot [7]. Water can be dropped from a dropper, pipette, or container. The capillary action draws the sample along the test strip and encounters the reagents in the conjugate pad. The sample flow arrow is indicated in a green arrow. If the target analyte is present, the water sample will then react with the reagents to produce a trapped complex in the detection zone. Trapping gives a visual indication, e.g., line or color band, of the presence of the analyte. A control line ensures that the assay is working correctly. If the target analyte is not present, no line (or a weaker line) will appear in the detection zone. The result depends upon the color production on the surface of the test strip, on the basis of which the quality of the water sample is assessed.
[0044] A core area with colorimetric or fluorescence detecting strips that show the results of the tests. Every test line is pre-coated with reagents for specific pathogens (e.g., E. coli, Vibrio cholerae, Giardia). Color reaction or fluorescence detects the presence of the target contaminant. Tests are read visually or imaged by a smartphone app. The function of the test strip is to show the result by visible change. The test strip comprises a series of individual lanes containing chemical reagents to identify a specific pathogenic bacterium. The presence of the pathogen triggers a chemical or biological reaction (e.g., a pH change, an enzyme-substrate reaction, or nucleic acid replication) that results in a color change or fluorescence. The results are read by eye or photographed using a smartphone and read on the computer.
[0045] Reference ladder: There is also a reference ladder, a graduated combined intensity scale on a test strip, and the detection area. It offers calibrated points of reference for quantitative analysis of signal intensity with allowance for illumination and smartphone camera variation. It is reproducible and precise across users and environments. It provides electronic or visual calibration. The paper strip is marked with solid printed fluorescence intensity bars or color scales near the test zone. It also allows smartphone applications and operators to calibrate the intensity of test results to known values to enable quantification. The benefits include adapting to environmental changes like light or camera performance.
[0046] Dotted chambers: Strategically designed permeable paper strips are utilized as fluidic delays. Sample flow rates are controlled in these regions to provide valuable time for definite reactions such as microbial lysis, nucleic acid cleaning, or immunologic capture. Timing becomes crucial in proper signal generation in the following regions. Its function is to control fluid timing. There are porous special structures printed or etched on paper to slow fluid movement, which offers sufficient time for biochemical reactions such as lysis of microbe (cell lysis), extraction or exposure to DNA/RNA and antigen, and pre-amplification incubation. The main outcome is that it offers multi-step processes without user intervention or electronics.
[0047] Waste Reservoir: A terminal fluid absorption pad Figure 4 [16] catches the expended water sample after completing the test course. It prevents backflow, provides no contamination hazard, and provides biosafety by safely holding potential infectious waste. Its main function is to hold and inactivate excess fluid and act as a sample reservoir when the test is complete. This is an absorbent tip at the strip's end, preventing contamination or reverse flow. It may contain antimicrobial chemicals to neutralize residual pathogens and offers biosafety when field-used and discarded.
[0048] Blister Reagent Pack: Small sealed packages are inserted within the strip with dry or lyophilized reagents (e.g., primers, enzymes, antibodies). They are finger-pressurizable or fluid-contact activatable and release contents into the flow stream to facilitate critical reactions like nucleic acid amplification or color development. Micro-compartments are placed strategically along the fluid path where important biochemical interactions occur. These are antigen-antibody recognition, enzyme-substrate catalysis, or isothermal amplification of DNA. The design facilitates immunoassay and molecular detection formats. Its main function is to dispense the required dry reagents at the right time. There are foil-sealed or membrane-sealed small packets incorporated in the test strip and, for activation, perforated by user finger press or pressure upon contact during flow.
[0049] The main contents are DNA primers, polymerases (for isothermal amplification), buffers or lysing agents, and antibodies for immunoassay. The main advantage is that it eliminates storage constraints, provides room-temperature stability, and increases shelf life.
[0050] Embedded Reaction Zones: Strategically designed permeable paper strips utilized as fluidic delays. Sample flow rates are controlled in these regions to provide valuable time for definite reactions such as microbial lysis, nucleic acid cleaning, or immunologic capture. The main function is that it enables a unique biochemical reaction., which is carried out by hermetically sealed vessels by layering or impregnating reagents in paper. The example reactions could be; antigen-antibody reaction (e.g., Lateral flow E. coli test), enzyme-substrate catalysis (e.g., detection of ammonia using urease), nucleic acid amplification (e.g., detection of DNA using LAMP). The design can include thermal insulation or reaction buffers with dry-state stabilization.
[0051] Colorimetric Calibration Ladder: A sequence of colored dots or gradient bars in a specified order is integrated into the strip. It offers onboard calibration reference via smartphone app or visual contrast. It reduces the interpretation error due to the user's subjectivity or vagueness of ambient light. It normalizes test interpretation. The color gradations of colored dots or calibrated color gradations to expected signal intensity levels. It aids users, or phone applications assess whether test signals indicate safe or dangerous contamination levels. Its design is scaled for use in different light conditions—daylight, cloud, and indoor.
[0052] Embedded Stripes and Chambers (Microfluidic Flow Paths): Paper-based architectures with hydrophobic and hydrophilic zones are required to guide sequential fluid flow in all functional regions (Figure 4, green arrow direction). Divided reaction space, signal formation, and reagent release are all coordinated within a confined system using patterned and stacked routes. Its function is to control and direct sample flow. The test strip is structured with hydrophilic channels and hydrophobic barriers (wax printing, laser etching, or photolithography), which guides the sample in sequence through detection, reaction, and calibration zones and protects from cross-reactivity of reagents.
[0053] It facilitates multiplexing analysis for many contaminants in one step. The main innovation is allowing dual-format molecular (DNA/RNA) and immunological testing in one test strip.
[0054] Multifunctional result readout is one of the greatest strengths of this platform. The results are readily observable to the naked eye visually by colorimetric markers in the zone of reaction, providing an instantaneous and simple observation of the test result. Likewise, the instrument is also provided as a smartphone app to quantify further or read out digitally. The software can capture an image of the test strip, read the intensity of color, and give a compact result as "Positive" or "Negative." The software can capture metadata like GPS location, time, and sample type for easy insertion into public health surveillance systems and real-time, simple epidemiology monitoring. Its ease, ruggedness, and speed render it a precious asset during outbreaks for mass screening or point-of-need diagnosis in resource-limited healthcare facilities or at the time of outbreak. Decentralized testing and enabling non-expert operation, a paper-based platform for enrichment bridges central gaps in infectious disease detection and brings necessary diagnostics to the point of need.
[0055] Paper biosensors constitute an up-and-coming and novel diagnostic tool, and all the more so for detecting pathogen nucleic acids in point-of-care and field conditions. Their capacity for quick, precise, and user-friendly measurements with minimal requirements on advanced laboratory equipment has made them of intense interest, particularly in low-resource settings and pandemic emergencies. LFAs are the most common platform of several paper-based biosensors because they are easy to use, less expensive, and versatile for many biological targets such as proteins, small molecules, and nucleic acids. Lateral flow assays are principle-based on the capillary action where the liquid test sample is propelled along a paper strip, typically in the form of a nitrocellulose membrane, without requiring an external power source or pumps. The standard LFA strip has some inherent components which are utilized together to analyze the sample and provide output. Components consist of a sample pad, conjugate pad, nitrocellulose membrane upon which detection lines are printed, and a terminal absorbent pad. The sample will start migrating down the strip after being applied to the sample pad. Buffers or surfactants will be on the sample pad to condition the sample and ensure good flow. The test sample then proceeds to the conjugate pad, interacting with dried reagents through labeled antibodies or nucleic acid probes such as gold nanoparticles or dyes such as colored latex beads. To enable LFAs to detect nucleic acids, a target RNA or DNA is chosen and identified using complementary probes, which become bound only in the presence of the genetic material of the pathogen organism. When the target is present in the sample, it complexes with the probe marker and forms a detectable complex, which will continue to flow along. When it arrives at the test region of the nitrocellulose membrane, the complex is stopped by bound secondary antibodies or probes. Visible line development, the cause of the positive test, is that which is formed. The control line confirms that the test is proper by attaching any excess conjugates in such a manner where the assay is correct. The form of the test enables the detection and semi-quantitation of analytes in terms of the intensity of lines.
[0056] LFAs have hence proved essential in several diagnostic uses, from infectious disease diagnosis to environmental sample collection, food protection, and genetic analysis. Their principal benefits are no or negligible pre-sample preparation, quick turnaround time, and room temperature stability and simple lay interpretation. This is why they can find potential applications in non-clinical environments like rural health centers, schools, refugee camps, and homes. Such development has further aided the integration of nucleic acid amplification techniques, such as isothermal amplification with LFAs, and also with increased sensitivity, further towards the detection of small amounts of pathogen RNA or DNA. LFA integration with smartphone-based testing is also gaining popularity, where digital readout, recording, and distance reporting to health authorities are enabled. Overall, LFAs represent the cornerstone pillar of paper-based biosensor science. Their portability and ease of use provide a foundation to democratize diagnosis and enable early infectious disease diagnosis at any point globally.
[0057] One innovation integrating point-of-care (POC) diagnostics with greater functionality, convenience, and portability is using paper analytical devices (PADs) in cell phones. The new integration provides the solution to the first-order requirement of low-cost, accurate, and decentralized diagnostic devices, especially for those diseases barring conventional laboratory devices. A good example of such integration includes developing a paper analytical device based on paper capable of performing dual-mode detection—fluorescence and colorimetric—of ß-glucosidase activity. It involves several biological processes and can be used as a disease biomarker. Because of this, detection needs to be effective and quick in clinical and environmental settings. The chip utilizes cellulose-based paper as a cheap and readily available substrate material that is highly well-established to be controllable by capillary fluid flow and bioreagent compatibility. Wax printing, a simple, low-cost, and high-throughput fabrication technique, is employed by the chip to fabricate the microfluidic channels of the paper. Hydrophobic barriers govern fluid sample and reagent flow along diverging paths to control reaction and interaction in targeted locations. It enables the device to perform multiple biochemical assay steps sequentially and reproducibly without sophisticated equipment and human expertise. When in use, the user deposits a liquid specimen onto the pre-scored sample zone on the device. The specimen is driven through microfluidic channels into reagent-infused spaces. The reagents selectively bind to ß-glucosidase upon detecting the presence of the enzyme in the sample. The enzymatic reaction leads to the detectability of a detectable change by two independent but complementary modalities: colorimetry and fluorescence. In colorimetric mode, the detection area is pigmented at visible wavelengths, which can be easily seen with the naked eye. Whereas the device in fluorescent mode, upon UV light excitation, will fluoresce if the enzyme acts optimally, the assay is still concentration-sensitive to analyte variation.
[0058] Besides quantification, the smartphone is incorporated into the paper-based sensor for broader availability. The smartphone is a generic platform that records the colorimetric or fluorescence signal as an image with its built-in camera. Advanced software programs on cell phones transform the images to determine the signal intensity and correlate it to the activity level of ß-glucosidase in the sample. It not only facilitates the collection and analysis of data but also includes geotagging, data logging, remote diagnosis, and real-time reporting to clinicians or data health repositories, with the integration of paper devices and mobiles being one of the success stories of the robust wave of digital health and decentralized diagnosis. It diminishes dependency on fixed labs and takes high-quality analytical ability to the site of need—home, field camps, rural clinics, or disaster relief. Dual-mode detection raises dependability by delivering instant visual confirmation supplemented by greater precision analytical measurement, decreasing false positives. Smartphone PADs such as the ß-glucosidase detector are a convergence of science made portable, mobile, and people-enabling design. They are revolutionizing data collection, analysis, and application in diagnosis to make healthcare more responsive, inclusive, and evidence-based.
[0059] The new paper-based immunoassay presents a low-cost, easy, and rapid alternative, particularly of utility for field testing and point-of-care testing. Immunoassay possesses a lateral flow nitrocellulose membrane substrate with ability to aid capillary migration of sample via pre-loaded test sites of test. Specific antibodies for the NS1 antigen treat the strip and, on a positive result, deliver a colored visible output. The density of subsequent color becomes strong with the rise in the concentration of the NS1 sample and contributes to naked-eye semi-quantitative visible interpretation. The better way that system is accomplished results from it possessing a smartphone app that achieves the highest possible precision and test convenience. Upon completing the assay, the participants take a picture of the test strip through the cell phone. The computer program then processes the image, measuring the color intensity using sophisticated image processing routines to compensate for extraneous light, camera, and other environmental factors. This yields less subjective, reproducible, and more accurate measurement of NS1 level compared to crude visual estimation. Mobile applications can also store and analyze information, and share with a simple button click. The test result can be tagged with metadata like time, where (with GPS), and patient, and is of greater usefulness for public health response and epidemiologic surveillance. It allows real-time mapping of hotspot clusters of infections and deployment of healthcare assets at the right time during outbreaks. Above all else, the system's portability and ease make it accessible so that even unknowledgeable CHWs or nonspecialists can employ it. Thus, its availability and penetration are significantly enhanced. The quantification-augmented and portable NS1 antigen-detector dengue paper-based immunoassay is a step towards democratizing diagnostics. It combines the low cost and ease of lateral flow assay with the benefit of digital technology to create a scalable, field-deployable, handheld device that will enhance dengue fever detection and control in most healthcare settings.
[0060] Paper-based microfluidic devices have also experienced explosive development in recent years, extending their applications beyond conventional clinical diagnostics to priority areas, including environmental monitoring. Maybe the most significant application here is to identify microbial contamination of food products and water—i.e., fecal contamination, a valuable biomarker for the possible presence of public health-relevant pathogens. One of the most significant achievements in the area has been developing a handheld paper-based biosensor system for real-time on-farm fecal contamination monitoring, especially for fresh produce farms where immediate and effective action is vital. A paper-based biosensor system has been developed as an off-the-shelf, fully integrated diagnostic kit with little training and no laboratory equipment. The product combines some of the most critical components into one package: a drop generator to facilitate sample preparation and handling, an imaging and heat module to facilitate and monitor biochemical reactions, and house paper-based biosensors as the detection substrate. These devices operate within a synergistic system together to yield sensitive and precise results in approximately an hour from sampling to readout. It starts with sampling the surface rinse samples of fruits and vegetables, which are then fed into the drop generator. The module offers reproducible and repeatable liquid sample deposition onto paper-based biosensors. The biosensors contain dry reagents, i.e., fluorometric or colorimetric substrates, which turn colored on being brought into contact with specific enzymatic activity or microbial markers in stools, e.g., Escherichia coli ß-glucuronidase. When they contact stools, the substrates turn colored or exhibit a fluorescent response, a clear, readable sign. The heating and imaging module will enhance reaction kinetics and biosensor sensitivity by providing optimal conditions for enzyme activity and high-resolution imaging.
[0061] Low-power operation is an everyday-use battery working as a field-and-outdoors-capable unit of suitable ruggedness. Images captured during assay time can be visually examined for real-time analysis or via a locally co-located smartphone program providing signal intensity and normalizing response reports. It is such a machine that comes well with promises to allow prompt decision-making among farmers in a way one will be able to determine the mode of contamination early enough such that the sale of toxic produce will be avoided, curtail the danger of foodborne sickness outbreaks, and allow conformation to food safety regulation. Above all, low costs and simplicity will make it feasible for poor-resource settings and small-scale farmers and producers who cannot afford lab facilities at the center. Its application is such that it will connect timely monitoring and diagnosis of environmental safety. By enabling in-situ on-site localized detection of fecal contamination, this paper-based microfluidic biosensor enables people to react immediately following contamination accidents, implement effective countermeasures promptly, and establish a safer food supply chain. Finally, these technologies identify the additional importance of paper-based diagnostics to ensure public health in clinical, agricultural, and environmental applications. These paper-based microfluidic device technologies, especially with smartphone technology, are a paradigm shift in environmental diagnostic capability, specifically in water quality diagnostic capability. The conventional pathogen diagnosis methods in water sources generally require high-technology laboratory equipment, expert technicians, and lengthy processing time, none of which are found in extensive rural or resource-limited areas. Paper microfluidic devices overcome all these limitations using inexpensive, portable, and simple modalities that are sensitive and specific.
[0062] Their most fascinating feature is the dual mode of detection: immunological and nucleic acid-based. The immunological mode of detection is an antibody-antigen reaction for detecting the presence of specific pathogens or fragments of pathogens. The immuno-based detection method is easy to prepare and is quick. Hence, it is best for rapid screening. However, nucleic acid detection like LAMP or CRISPR-based assays would be able to identify genetic markers of target pathogens. The genetic methods are several orders of magnitude more discriminative and sensitive, with the capability of identifying trace amounts of pathogens that fall below the detection level when analyzed by conventional assays. Paper platform integration with such methodologies has several benefits. Paper as a platform is inexpensive, lightweight, and biodegradable, enabling capillary-driven fluid movement without needing an outside power source. It thus makes the whole system cost-effective and sustainable. Other than that, paper-based systems also lend themselves well to being very easily mass-produced and, therefore, production costs can be proportionally minimal, making mass deployment easily possible through mass outbreaks in public health programs or in a crisis management scenario.
[0063] Smartphone connectivity further adds to the device's amenity and convenience. Since they are equipped with cameras and sensors, smartphones can quantify colorimetric or fluorescence from paper-based assays, quantify amounts, and transmit data to cloud storage or public health authorities in real time for surveillance. All tests through this connection will more likely be an instrument point of data in a large surveillance web, feeding data into early warning systems and evidence-based public health interventions. Besides, the machines can assist in conducting the walk test through steps that reduce error and require special training. With ease of mobility and fast turnaround time, the machines can easily find applications in rural regions far away from hospitals and during natural disasters or humanitarian crises when there is a need for clean water. These are the types of circumstances where the capability to determine in seconds and with accuracy whether water is safe can be a matter of life and death. Beyond pathogens, research even goes as far as to allow such devices to enhance their capability for detecting chemical contaminants such as heavy metals, pesticides, and endocrine disruptors to give a more complete characterization of water quality. The union of paper microfluidic devices with cell phone technology is thus revolutionizing water analysis treatment. Besides giving individuals access to more potentially life-saving diagnosis information, it enables communities to monitor and safeguard their waters.
[0064] Sustained innovation and mass adoption of new water-testing technology, including paper microfluidic devices paired with cellular phone capability, could be critical in global efforts to combat waterborne disease. These diseases, having been ingested by inciting pathogens like bacteria, viruses, and protozoa of the contaminated drinking water, remain a leading cause of morbidity and mortality, most notably within poor-income and resource-poor settings. Current water analysis techniques, as practical as they are, are inadequate in such settings since they are expensive, machine-based, and labor-intensive. Field-portable, easy-to-use, and inexpensive diagnostic kits can transform the scene. Third-generation instruments facilitate public intervention by enabling rapid and reproducible detection of microbial contaminants. Relative to their traditional lab-based counterparts, which would take days to provide data, point-of-use instruments can provide decision-enabling information within hours or minutes. Rapid feedback is the central tenet facilitating quick action, ranging from imposition boil-water advisories and disinfection protocols to quarantining source contaminants. Premature contamination detection prevents outbreaks, reduces transmission rates, and reduces the magnitude of public health crises. It also allows for the simplicity of public health responses at the community level. The devices are simple to train on, and neighbors or local government employees can use them daily to test for water quality. Smartphones allow for improved performance and efficiency of the devices since they allow for digital presentation, software interpretation of data, and geotagging of pollution events. Mobile phone information can be relayed instantly to central data stores, from which governments and public health authorities can monitor trends, hotspot detection, and enhanced resource allocation. In at-risk groups like children, older adults, immunocompromised persons, and slum/resettlement community residents, these phones are a significant first line of defense. The availability of safe drinking water in these communities is usually the primary infrastructural and logistical barrier. Low-cost point-of-care diagnostics can circumvent much of the traditional constraints and provide life-saving data where it is most desperately needed. They have a direct role in reducing disease burden, healthcare costs, and quality of life.
[0065] Moreover, applications of these technologies enable long-term global environmental and health goals, such as United Nations Sustainable Development Goal 6: universal access to water and sanitation. Employed in water management, these technologies can help improve systematic water resilience and security. Therefore, additional research and application of innovative paper-based water testing technology are a feasible and attainable step towards preventing the onset of world waterborne disease. By the in-situ, real-time detection of microbial risk, the devices increase public health infrastructure's data-loudness and responsiveness and make clean drinking water accessible even to marginalized communities. Briefly, the integration of paper microfluidics and smartphone platforms is a paradigm shift towards field-deployable water analysis. The novel merger breaks through most of the conventional disadvantages of the conventional lab-based analysis, including high operation costs, highly skilled human capital, dependency on extremely advanced hardware, and delay in retrieving sample reports. The aforementioned limitations have hitherto limited the quantification of water quality in real-time, particularly in geographically remote, poor-resource, or disaster-stricken regions. Mobile technology-enabled paper-based diagnostics present a rational, cost-effective, and scalable remedy to such demand. Paper microfluidic devices are developed to execute advanced biochemical assays using tiny amounts of reagent and fluid in a disposable, biodegradable, and portable package. They are designed so perfectly to generate and disseminate that they are the optimal choice to use in the field where installation in a laboratory is not possible or impracticable. The technology can identify a wide range of pens considered contaminants, ranging from microbial pathogens to heavy metals and chemical toxins, giving an accurate impression of water quality.
[0066] Connectivity with smartphones enhances the diagnostic strength of such devices in several important areas. Second, cell phones allow for imaging, inspection, and recording of the visual output of the assays—e.g., colorimetric or fluorescence signals—through some apps. Applications can subsequently utilize in-built algorithms to examine data and provide users with easily understandable, real-time feedback. GPS and connectivity are also facilitating secondly, replication of test results and assigning them location tags into core databases, thereby making it possible to track and map contamination events in real time. The electronic interfaces are playing important roles in ensuring timely public health interventions and intervention planning over the long term for safe water and resource management.
[0067] Future developments in material science, biosensor technology, and microcomputer miniaturization will further improve the sensitivity, specificity, and shelf-life of such integrated systems. There must also be space for emerging technologies such as multiplexed testing capability (to test for two or more contaminants), auto-sustaining paper-based sensors, and machine-based reading for higher precision and easy-to-follow instruction. They will expand the uses of the water test kit to become a standard tool of environmental monitoring, disaster response intervention, and intervention in public health. Second, their broader implications converge with global efforts toward universal clean water access on terms that the United Nations Sustainable Development Goals dictate. Direct access to responsive and reliable water quality sensing and phone-readable paper-based microfluidics enables individuals to be lord and master in their own home as water resource managers and enables local informed decision-making to emerge in place. These are, however, more of a paradigm shift in water safety delivery and strategy and less an issue of technology development. With every step in this R&D being taken, these technologies can contribute more towards protecting public health, establishing environmental resilience, and providing global water security.
[0068] The disclosure has been described with reference to the accompanying embodiments herein and the various features and advantageous details thereof are explained with reference to the non-limiting embodiments in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein.
[0069] The foregoing description of the specific embodiments so fully revealed the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the scope of the embodiments as described herein.
, C , Claims:We Claim:
1) A handheld diagnostic device for assessing the quality of a water sample, comprising:
- a test strip slot configured to receive a nitrocellulose or paper-based test strip;
- grooves for securely positioning said test strip;
- a water sample loading tray slot to introduce the water sample; and
- an ultraviolet (UV) light source operable via battery power to facilitate detection of fluorescence signal from said test strip;
wherein the test strip comprises microfluidic channels that enable unidirectional capillary flow of said sample to detection zones pre-loaded with analyte-specific reagents.
2) The diagnostic device as claimed in claim 1, wherein the paper-based test strip comprising:
- a sample loader and distributor zone;
- microfluidic capillary channels enabling uniform sample flow;
- at least one detection line coated with reagents for specific target analytes;
- a control line for process validation; and
- an absorbent waste reservoir for collecting expended sample,
wherein said test strip further comprises embedded blister reagent packs and porous fluidic delay chambers for timed biochemical reactions.
3) The diagnostic device as claimed in claim 1, wherein the UV light is positioned directly above the test strip slot, such that when activated, it induces visible fluorescence on the test strip for detection of target analytes not observable under natural light.
4) The diagnostic device as claimed in claim 1, wherein the blister reagent packs are finger-pressurizable or activated upon fluid contact to release lyophilized or dried reagents selected from a group comprising antibodies, primers, enzymes, and colorimetric substrates.
5) The diagnostic device as claimed in claim 2, wherein the porous fluidic delay chambers provide controlled dwell time for reactions including but not limited to microbial lysis, nucleic acid release, and antigen-antibody binding.
6) The diagnostic device as claimed in claim 1, wherein a reference intensity ladder or colorimetric calibration zone is printed adjacent to the test zone for visual or digital quantification of the signal intensity.
7) The diagnostic device as claimed in claim 1, wherein the device is configured for visual interpretation by the naked eye or integrated with a smartphone application for digital imaging, signal processing, and result readout.
8) The diagnostic device as claimed in claim 1, wherein the smartphone application is operable to record fluorescence or colorimetric data, perform signal quantification, geotag the result, and transmit said data to a remote server or public health database.
9) The diagnostic device as claimed in claim 2, wherein the hydrophilic and hydrophobic zones are patterned using wax printing, laser etching, or photolithography to guide the fluid flow sequentially through reaction, detection, and calibration regions and prevent cross-contamination between reagents