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Nitrocellulose Membranes for Lateral Flow Tests: A Guide to Choosing the Right Membrane for Development and Manufacturing
Lateral flow tests have become a ubiquitous tool in various industries, enabling rapid and reliable on-site diagnostics. At the heart of these tests lies the nitrocellulose (NC) membrane, a critical component that plays a pivotal role in capturing and detecting the target analytes. As a consultant in the field of lateral flow (LF) tests, I understand the importance of selecting the right membrane during development and manufacturing and can guide you to ensure optimal performance and accuracy.
“When I was working as head of manufacturing in a small biotech company I was send for training (and networking) to a nice workshop in Ireland hosted by one of the top NC manufacturers. I produced there also some HCG lateral flow tests and was very proud of myself. However, as I came home and showed those “masterpieces” to my LF chief technician, he just rolled his eyes and told me, that I will never be allowed to work in LF manufacturing, since I scratched the membrane with my nails…I just wanted to be super accurate – and killed the heart of the LF strip… “
In this post, we will explore the world of nitrocellulose membranes, examining the key considerations and factors to keep in mind when choosing the most suitable membrane for your lateral flow test applications. We will explore the complexities of membrane properties, coating techniques, modifications, and quality control measures that contribute to the overall success of lateral flow tests.
1. Introduction to Nitrocellulose Membranes:
Nitrocellulose membranes are the core part of lateral flow tests, providing the surface for for capturing and detecting target analytes in various applications, including medical diagnostics, environmental monitoring, and food safety testing. Their intrinsic properties – 1) enabling a lateral flow of a liquid and 2) easy adsorption of proteins – make them ideal for use in point of care applications.
Nitrocellulose is a highly porous and biocompatible material. It is derived from cellulose, a natural polymer found in plant cell walls, by treating it with nitric acid. This process results in the formation of a fibrous network of highly interconnected pores within the membrane structure. The pore size of nitrocellulose membranes can be precisely controlled during the manufacturing process, allowing customization to meet specific performance requirements.
The capillary action of the membrane allows for rapid movement of liquid samples, facilitating the migration of analytes, reagents and detection molecules through the test zones. This attribute is important for achieving quick and efficient lateral flow test results.
In addition to their porosity and flow properties, nitrocellulose membranes offer a high binding capacity for proteins. The surfaces of these membranes can be modified to enhance specific interactions. This customization enables efficient and selective analyte capture, ensuring sensitive and reliable detection in lateral flow assays.
Another physical property of nitrocellulose membranes to consider is their thickness. Membrane thickness affects the sample volume needed for reliable test performance. [1, 2, 3, 4, 5, 6, 7, 8, 9]
“Do you know how LF membranes are produced? Read on, we will come to this point and its implications later.”
2. Factors to Consider when Choosing Nitrocellulose Membranes:
When it comes to selecting nitrocellulose membranes for lateral flow assays, several important factors need to be considered to ensure optimal assay performance and reliable results. The following factors should be taken into account:
- Pore Size and Flow Rate:
The pore size of the nitrocellulose membrane plays an important role in controlling the flow rate and the ability to capture and transport analytes. Smaller pore sizes provide higher resolution and sensitivity due to slower flow but may impede the flow of larger analytes or aggregates and can extend unspecific binding. Pores have a size of 3-20 µm for the typically employed membrane types.
- Material Thickness:
The thickness of the nitrocellulose membrane influences the volume of sample required for effective detection. Thicker membranes can accommodate larger sample volumes, allowing for better detection sensitivity, but may require longer assay times. Additionally, the gain in sensitivity might be hampered depending on the pre-treatment of the membrane and immobilization of capture molecule as the detection particles can most often only be detected from the topmost few µm of membrane. Thinner membranes, on the other hand, can reduce the required sample volume but may sacrifice sensitivity. Balancing these considerations is crucial to meet the specific requirements of the lateral flow test. Additionally, also width of the dispensed lines, and therefore visibility of test results is influenced to some degree by the thickness, too. Furthermore, the handling during manufacturing is depending on the thickness – at least for unbacked membranes, that is pure nitrocellulose without polyester support.
- Surface Chemistry and Binding Capacity:
Nitrocellulose membranes can be modified with different surface chemistries to enhance specific interactions with target molecules. These modifications can include functional groups, coatings, or immobilized affinity ligands. The choice of surface chemistry should be carefully considered based on the nature of the analyte and the desired sensitivity and specificity of the assay. Additionally, the binding capacity of the membrane, which refers to its ability to immobilize capture agents, is a critical factor for efficient analyte capture and detection.
- Sample Matrix Compatibility:
Consideration should be given to the type of sample matrix in which the lateral flow test will be performed. Certain sample matrices, such as whole blood or highly viscous fluids, may require membranes with specific properties, such as enhanced wicking capabilities or resistance to interference from matrix components. Understanding the sample matrix and its potential impact on the performance of the assay is a good basis for selecting the appropriate nitrocellulose membrane.
By carefully considering these factors during the selection of nitrocellulose membranes - especially pore size and flow rate – developers and manufacturers of lateral flow tests can ensure optimal assay performance, sensitivity, and specificity for their distinct applications. [1, 2, 4, 9, 10]
“Did you know that the wicking rate of the membranes is specified by the manufacturers in the quite awkward unit of seconds per 4 cm?”
3. Membrane Coating Techniques:
The coating of nitrocellulose membranes is a critical step in the manufacturing process of lateral flow tests. Various coating techniques are employed to ensure uniform and precise deposition of capture agents, such as antibodies or antigens, onto the membrane surface. The choice of coating technique can significantly impact the performance and reliability of the lateral flow assay.
- Dispensing:
This is the most common technique for immobilization of the capturing reagents. The liquid is distributed with a precise and continuous flow of some µL per cm at the respective position of the test- and/or control line. This allows for controlled and reproducible application of the proteins and minimizes the risk of oversaturation or uneven distribution, resulting in consistent and reliable performance of the assay.
- Dip Coating:
Dip coating is a widely employed technique for blocking of the surface. It is mostly performed after immobilization of the capturing molecules. The nitrocellulose membrane is immersed in a solution containing the blocking reagent. Capillary action draws the solution into the pores of the membrane, facilitating the deposition of the blocker. Dip coating is relatively simple and cost-effective, making it a popular choice for many lateral flow test manufacturers.
- Spray Coating:
- Spray coating involves the use of a spray nozzle to disperse the capture agent or blocking solution onto the nitrocellulose membrane. It can be used to cover a wide area as well as thin line – depending on the setup of the spray cone. This technique allows for more precise control over the coating process, ensuring an almost uniform distribution of the reagent across the membrane surface but without wasting too much solution as for dip coating. Spray coating is particularly useful for large-scale production, where high throughput, efficient use of reagents and consistent coating quality are essential.
Each coating technique has its advantages and considerations. Factors such as coating uniformity, reproducibility, speed, and scalability should be evaluated when selecting the most suitable technique for a particular lateral flow test. Additionally, optimization of the coating solution composition, including the concentration of the capture agent and additives, can further enhance the performance of the coated nitrocellulose membrane. Finally, the coating process can also damage the membrane when using the wrong machine parameters. So careful optimization is also necessary here.
By employing the appropriate coating technique and optimizing the coating process, manufacturers can ensure the consistent and reliable deposition of capture agents on nitrocellulose membranes, thereby improving the sensitivity and specificity of lateral flow tests.
One has also to keep in mind that there are certain room conditions before, during and after coating of the membrane – namely, humidity and temperature – that have a not to be underestimated effect on the coating efficiency and assay performance. Those conditions and parameters ideally must be controlled, monitored and optimized. [1, 10, 11]
4. Membrane Modifications and Functionalities:
Membrane modifications can play an important role in optimizing the performance of nitrocellulose membranes used in lateral flow tests. These modifications can introduce additional functionalities and improve the sensitivity, stability, and specificity of the assays.
- Surface Blocking:
Surface blocking is a common modification that involves the application of blocking agents, such as proteins or surfactants, onto the membrane surface. This step helps prevent nonspecific binding of unwanted substances and reduces background noise in the assay. Surface blocking enhances the specificity of the lateral flow test by minimizing false-positive or false-negative results.
- Stabilizers:
Incorporating stabilizers during coating can provide additional functionalities. Stabilizing agents, for example sugars, can improve the stability and shelf life of the immobilized proteins, maintaining the activity by preventing degradation or loss of performance over time.
- Hydrophilic/Hydrophobic Modifications:
Modifying the membrane's hydrophilicity or hydrophobicity can influence the sample flow and interaction with the analyte. Hydrophilic modifications can enhance wicking properties, promoting rapid and uniform flow of the sample across the membrane. Hydrophobic modifications can be useful for containing reagents or controlling the flow rate. Tailoring the hydrophilic or hydrophobic properties of the membrane can improve assay performance.
- Surface Activation:
This is a bit of a special topic and mostly not used for “standard” lateral flow tests. Surface activation involves treating the nitrocellulose membrane to enhance its reactivity and enable covalent binding of biomolecules. Surface activation methods include plasma treatment, chemical modification, or physical techniques like UV irradiation. Activated membranes can offer increased binding capacity and stability for immobilizing capture agents, improving the overall performance of the lateral flow test – albeit increasing manufacturing effort.
By carefully considering these membrane modifications and functionalities, lateral flow test developers can tailor the nitrocellulose membranes to their specific assay requirements. These modifications optimize the sensitivity, stability, and reliability of the test, ensuring accurate and consistent results. [1, 10, 11, 12]
5. Quality Control and Testing:
Ensuring the quality and reliability of nitrocellulose membranes used in lateral flow tests is paramount to achieving accurate and consistent results. Well established quality control (QC) processes and testing protocols are vital throughout the manufacturing process.
- Raw Material Evaluation:
Thorough evaluation of the raw materials, especially the nitrocellulose, is important to ensure their suitability for LF production. The physical characteristics of the raw material such as correct dimension, flow rate and homogeneity must meet specifications to maintain the desired performance of the final product. It is worthwhile to notice here that nitrocellulose is produced in processes similar to paper. That is, large mother rolls of several meters width and several hundreds of meters of length. 100% homogeneity for each and every special application cannot be guaranteed by the membrane manufacturers. It is therefore the task of the LF manufacturer to identify suitable raw material for his/her processes: A meaningful incoming goods process is necessary.
- Process Validation:
Validating the membrane coating and assembly manufacturing process is essential for consistency and reproducibility. Process parameters, such as room conditions, dispensing/dipping/spraying parameters, drying conditions (and in a later process-step also curing time), need to be optimized and validated to ensure consistent performance across batches.
- Performance Testing:
Performance testing involves evaluating the key characteristics of the coated nitrocellulose membranes, such as sensitivity, specificity, and limit of detection, homogeneity of the lines and others. Validated test analytes or simulated samples can be used to assess the membrane's performance against established criteria. Statistical analysis and comparison to reference materials or known standards can aid in determining the membrane's accuracy and reliability.
- Stability Testing:
Stability testing is key to assess the shelf life and long-term performance of the nitrocellulose membranes. Accelerated aging studies, storage condition evaluations, and real-time stability assessments help ensure that the membranes - raw material as well as coated intermediate products – maintain their functionality and performance over time.
- Quality Assurance Documentation:
Proper documentation of quality control processes, test results, and specifications is essential for traceability and compliance. Well-documented quality assurance records facilitate transparency and enable efficient troubleshooting and batch-to-batch consistency.
“In my opinion implementing robust quality control and testing procedures throughout the manufacturing process is fundamental to deliver high-quality lateral flow tests. The material itself is produced by the highest standard in industrial quality, but it is per definition a not homogeneous material. Additionally, each and every membrane manufacturer adds a not disclosed mixture of certain chemicals and surfactants to the nitrocellulose to render the final material more hydrophilic – otherwise your lateral flow test might not even start to flow when adding the sample.”
By adhering to strict quality standards and continuous process improvement, manufacturers can ensure the reliability and performance of their membranes, ultimately contributing to the accuracy and trustworthiness of lateral flow test results. [11]
“Did you know that the German balance manufacturing company Sartorius was the first to manufacture nitrocellulose industrially in 1960?”
6. Future Developments and Outlook:
As the field of lateral flow testing continues to advance, there are several exciting developments and future prospects for nitrocellulose membranes – also not only limited to NC inherent but complementing technologies:
- Integration of Smart Technologies:
The integration of smart technologies, such as smartphone-based detection and data analysis, holds great potential for lateral flow tests. By leveraging the capabilities of smartphones, including built-in cameras and image processing algorithms, the interpretation and quantification of lateral flow test results can be automated, standardized and made more robust against inhomogeneity of the membrane material. This will enhance result accuracy, data management, and remote monitoring capabilities, expanding the applications of lateral flow tests.
- Enhanced Sensitivity:
Ways to enhance the sensitivity of lateral flow tests are explored by improving signal amplification methods and optimizing the membrane structure. This includes incorporating novel nanomaterials, such as nanoparticles or nanocomposites, to enhance the signal generation and improve the limit of detection. These advancements may enable the detection of low-abundance analytes and increase the overall sensitivity of lateral flow assays.
- Multiplexing Capabilities:
Multiplexing, the ability to detect multiple analytes simultaneously, is an area of significant interest in lateral flow testing. Efforts are being made to develop nitrocellulose membranes that can accommodate multiple test lines, each targeting a specific analyte. This enables more comprehensive and efficient testing, saving time and resources. Researchers are exploring innovative techniques such as spatially encoded particles, microfluidics, or digital imaging to enable multiplexing on a single membrane.
- Customization and Tailored Designs:
With advancements in manufacturing techniques, there is an increasing trend towards customized and tailored designs of nitrocellulose membranes. Researchers are exploring methods to fabricate membranes with specific characteristics, to meet the requirements of specific assays or target analytes. Customized membranes such as flow-modified or flow-controlled will allow for optimized performance, improved sensitivity, and better compatibility with different lateral flow test formats.
- Sustainability:
The focus on sustainability in lateral flow testing is growing, and future developments aim to address environmental concerns. Researchers are exploring the use of eco-friendly materials and manufacturing processes, such as bio-based polymers and water-based coatings, to reduce the environmental footprint of nitrocellulose membranes. Additionally, efforts are being made to optimize production efficiency, minimize waste generation, and develop biodegradable or recyclable lateral flow test devices. By prioritizing sustainability, lateral flow testing can contribute to environmentally conscious practices and align with global efforts towards a greener future.
Future developments hold great promise for the continued evolution of nitrocellulose membranes in lateral flow tests. With ongoing research and technological advancements, we can expect improved and more user-friendly point-of-care applications. [13, 14, 15, 16, 17, 18, 19, 20]
In conclusion, nitrocellulose membranes are the core of every lateral flow tests, these provide the surface for detection of the analyte. Selecting the right membrane is one key for optimal performance and accuracy. Considerations include pore size, material thickness, surface chemistry, and coating techniques. Membrane modifications can enhance sensitivity, stability, and specificity. Quality control measures ensure reliable results through raw material evaluation, process validation, performance and stability testing, and quality assurance documentation. Future developments focus on enhancing sensitivity, multiplexing capabilities, smart technology support, customization, and sustainability. Exciting advancements will drive the evolution of (nitro)cellulose membranes in lateral flow tests, providing improved sensitivity, user-friendly applications and sustainability. Imagine lateral flow tests, that could just be regarded as biodegradable waste – instead of residual waste used only for “energetic recovery”.
“Researchers estimate the UK's lateral flow testing programme has produced enough plastic waste to fill 200,000 bathtubs or 19 Olympic swimming pools.”[21]
If you like to discuss about choice of lateral flow membrane in more detail, don’t hesitate to contact me and book a meeting.
Share your own experiences, ideas, or challenges related to lateral flow test development and manufacturing and send me a message.
References:
[1] https://www.cytivalifesciences.com/en/us/solutions/lab-filtration/knowledge-center/Considerations-for-membrane-selection-for-lateral-flow-immunoassays - 15-Jun-2023.
[2] https://www.sartorius.com/en/products/oem/oem-membranes-and-devices/diagnostic-membranes/unisart-lateral-flow - 15-Jun-2023.
[3] https://link.springer.com/chapter/10.1007/978-1-59259-951-6_4 - 15-Jun-2023.
[4] https://link.springer.com/chapter/10.1007/978-1-59745-240-3_6 - 15-Jun-2023.
[5] Deng, Y., Jiang, H., Li, X. et al. Recent advances in sensitivity enhancement for lateral flow assay. Microchim Acta 188, 379 (2021).
[6] Amadeo Sena-Torralba, Ruslan Álvarez-Diduk, Claudio Parolo, Andrew Piper, and Arben Merkoçi, Toward Next Generation Lateral Flow Assays: Integration of Nanomaterials, Chemical Reviews 2022 122 (18), 14881-14910, DOI: 10.1021/acs.chemrev.1c01012.
[7] Koczula KM, Gallotta A., Lateral flow assays. Essays in Biochemistry. 2016 Jun;60(1):111-120.
[8] Hsiao, W.W.-W.; Le, T.-N.; Pham, D.M.; et al., Recent Advances in Novel Lateral Flow Technologies for Detection of COVID-19. Biosensors 2021, 11, 295.
[9] https://www.fortislife.com/nitrocellulose-membrane-selection - 15-Jun-2023.
[10] Application Note Millipore: Performance of Estapor® Microspheres and Hi-Flow™ Plus Membranes in a Lateral Flow Assay for Human Chorionic Gonadotropin (hCG) (https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/108/650/an1222en00-mk.pdf)
[11] Sukumaran A, Thomas T, Thomas R, Thomas RE, Paul JK, Vasudevan DM. Development and Troubleshooting in Lateral Flow Immunochromatography Assays. Indian J Clin Biochem. 2021 Apr;36(2):208-212.
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[13] Muhammad Sajid, Abdel-Nasser Kawde, Muhammad Daud, Designs, formats and applications of lateral flow assay: A literature review, Journal of Saudi Chemical Society, Volume 19, Issue 6, 2015, Pages 689-705.
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[15] Lin, D., Li, B., Fu, L. et al. A novel polymer-based nitrocellulose platform for implementing a multiplexed microfluidic paper-based enzyme-linked immunosorbent assay. Microsyst Nanoeng 8, 53 (2022).
[16] Ruihua Tang, Ming Yue Xie, Min Li, Lei Cao, Shangsheng Feng, Zedong Li, Feng Xu, Nitrocellulose Membrane for Paper-based Biosensor, Applied Materials Today, Volume 26, 2022, 101305, ISSN 2352-9407.
[17] Quesada-González D, Stefani C, González I, de la Escosura-Muñiz A, Domingo N, Mutjé P, Merkoçi A. Signal enhancement on gold nanoparticle-based lateral flow tests using cellulose nanofibers. Biosens Bioelectron. 2019 Sep 15;141:111407.
[18] Yew CHT, Azari P, Choi JR, Muhamad F, Pingguan-Murphy B. Electrospun Polycaprolactone Nanofibers as a Reaction Membrane for Lateral Flow Assay. Polymers (Basel). 2018 Dec 14;10(12):1387.
[19] Iles AH, He PJW, Katis IN, Horak P, Eason RW, Sones CL. Optimization of flow path parameters for enhanced sensitivity lateral flow devices. Talanta. 2022 Oct 1;248:123579.
[20] Xue Wang,a Chao-Hua Xue, ORCID logo *ab Dong Yang,*c Shun-Tian Jia,b Ya-Ru Ding,b Lei Lei,c Ke-Yi Gaoc and Tong-Tong Jiac, Modification of a nitrocellulose membrane with nanofibers for sensitivity enhancement in lateral flow test strips, RSC Adv., 2021, 11, 26493
[21] https://www.bbc.com/news/av/health-60929956 - 15-Jun-2023
