A device architecture for three-dimensional, patterned paper immunoassays.

The correct interpretation of the result from a point-of-care device is crucial for an accurate and rapid diagnosis to guide subsequent treatment. Lateral flow tests (LFTs) use a well-established format that was designed to simplify the user experience. However, the LFT device architecture is inherently limited to detecting analytes that can be captured by molecular recognition. Microfluidic paper-based analytical devices (μPADs), like LFTs, have the potential to be used in diagnostic applications at the point of care. However, μPADs have not gained significant traction outside of academic laboratories, in part, because they have often demonstrated a lack of homogeneous shape or color in signal outputs, which consequently can lead to inaccurate interpretation of results by users. Here, we demonstrate a new class of μPADs that form colorimetric signals at the interfaces of converging liquid fronts (i.e., lines) to control where colorimetric signals are formed without relying on capture techniques. We demonstrate our approach by developing assays for three classes of analytes-an ion, an enzyme, and a small molecule-to measure using iron(III), acetylcholinesterase, and lactate, respectively. Additionally, we show these devices have the potential to support multiplexed assays by generating multiple lines in a common readout zone. These results highlight the ability of this new paper-based device architecture to aid the interpretation of assays that create soluble products by using flow to constrain those colorimetric products in a familiar, line-format output.

Cross reactivity of serological response to SARS-CoV-2 vaccination with viral variants of concern detected by lateral flow immunoassays

Since the pandemic outbreak of Covid-19 in December 2019, several lateral flow assay (LFA) devices were developed to enable the constant monitoring of regional and global infection processes. Additionally, innumerable lateral flow test devices are frequently used for determination of different clinical parameters, food safety, and environmental factors. Since common LFAs rely on non-biodegradable nitrocellulose membranes, we focused on their replacement by cellulose-composed, biodegradable papers. We report the development of cellulose paper-based lateral flow immunoassays using a carbohydrate-binding module-fused to detection antibodies. Studies regarding the protein binding capacity and potential protein wash-off effects on cellulose paper demonstrated a 2.7-fold protein binding capacity of CBM-fused antibody fragments compared to the sole antibody fragment. Furthermore, this strategy improved the spatial retention of CBM-fused detection antibodies to the test area, which resulted in an enhanced sensitivity and improved overall LFA-performance compared to the naked detection antibody. CBM-assisted antibodies were validated by implementation into two model lateral flow test devices (pregnancy detection and the detection of SARS-CoV-2 specific antibodies). The CBM-assisted pregnancy LFA demonstrated sensitive detection of human gonadotropin (hCG) in synthetic urine and the CBM-assisted Covid-19 antibody LFA was able to detect SARS-CoV-2 specific antibodies present in serum. Our findings pave the way to the more frequent use of cellulose-based papers instead of nitrocellulose in LFA devices and thus potentially improve the sustainability in the field of POC diagnostics.

Utility of saliva in rapid post-mortem rabies diagnosis in animals by lateral flow assay.

Point-of-care diagnostic (POC) is of utmost importance for the fight against pandemics which provides easy, instant results right at your place. POC can transform healthcare management and create superior infrastructure. In POC, employing lateral flow assays (LFAs) can accelerate the assessment and shorten the treatment time. LFA is a small handheld device made from paper that controllably delivers the analyte without the need of an active pump to the detection area to produce the signal, generally coloured nanoparticles (Au, carbon).Conventional LFA device is made of four components, sample pad, conjugate pad, nitrocellulose membrane and absorbent pad arranged sequentially. At the sample pad, the analyte is dispensed. From here, the analyte moves to the conjugate pad and hybridizes with recognition antibodies (placed onto nanoparticles) to form analyte-antibody-gold nanoparticles conjugate. Further movement takes this to the nitrocellulose membrane. Here, other recognizant antibodies are placed at the test and control line to immobilize by forming the sandwich assay with the analyte-antibody-gold nanoparticles. The excess liquid is absorbed in the absorbent pad. From these components of the LFA device, nitrocellulose membrane and antibodies need replacements as they increase the device's overall cost. Cellulose paper holds promise to counter nitrocellulose membrane; however, the development of fabrication methods for cellulose paper-based LFA devices is needed. In comparison to antibodies, aptamers improve batch-to-batch variability, ruggedness, and longevity of LFA devices.This work describes the fabrication of half-strip cellulose paper (Whatman® Grade 4 filter paper) based LFA device exploiting aptamers as the recognition element for antigen detection. The covalent bonding of aptamers was achieved by NanoCheck-ATH® (NC) and p-phenylene disothiocyanate (PDITC). NC provides plentiful amine functional groups and constricts the test line width by covalently bonding aptamer to the NC region only. The chitosan-based NanoCheck on paper assists in realizing sharp test and control lines by providing the controlled surface for PDITC reaction through amine groups. PDITC functions as a linker between NC and aptamers. Finally, a proof-of-concept nucleocapsid protein of SARS-CoV-2 as an antigen detection was shown with the minimal detectable concentration down to ~20 ng/mL with excellent selectivity towards casein, BSA, and albumin. We envisage that using facile modification chemistry of NC, PDITC, and aptamers for antigen detection will allow the fabrication of paper-based LFA devices in a roll-to-roll setting and, thus, decrease the overall cost. The developed method is generic and can be adapted to detect other antigens to fight future pandemics.

Automation Highlights from the Literature

Lateral flow assays (LFAs) are immunochromatographic point-of-care devices that have greatly impacted disease diagnosis through their rapid, inexpensive, and easy-to-use form factor. While LFAs have been successful as field-deployable tools, they have a relatively poor limit of detection when compared to more complex methods. Moreover, most design and manufacturing optimization is achieved through time- and resource-intensive brute-force optimization. Despite increased interests in LFA manufacturing, more quantitative tools are needed to study current manufacturing protocols and therefore, optimize and streamline development of these devices further. In this work, we focus on a critical LFA component, colloidal gold conjugated to a detection antibody, one of the most commonly used reporter elements. This study utilizes inductively coupled plasma optical emission spectroscopy (ICP-OES) in conjunction with a lateral flow reader to quantitatively analyze colloidal gold distributions at the read-out test and control lines, as well as residual gold on the conjugate pad and other flow through regions. Our goals are to develop a more rigorous understanding of current LFA designs as well as a quantitative understanding of shortcomings of operational characteristics for future improvement. To our knowledge, this is the first time that ICP-OES has been used to study the initial distribution of colloidal gold on an unused LFA and its redistribution after a test is performed. Using three different brands of commercially available malaria LFAs, gold content was measured within each section of an LFA at varying parasite test concentrations. As expected, the total mass of gold remained unchanged after LFA use; however, the total mass of initial gold and its redistribution varied among manufacturers. Importantly, there are also some inherent inefficiencies that exist in these commercial LFA designs; for example, only 30% of the total gold deposited onto Brand A LFAs binds to the test and control lines, sections of the test that contain interpretable signal. Using information gathered with this method, future devices could be more purposefully engineered to focus on improved binding efficiency, resulting in reduced costs, improved limit of detection, and diminished test-to-test and manufacturer-to-manufacturer variability.

Although lateral flow tests (LFTs) are easy-to-use diagnostics, they have fundamental limitations for sequential multistep assay that can be reduced to a single chemical reaction step. Paper-based microfluidic devices have attracted considerable attention for use in automatic multi-step assays because paper can be an excellent platform to control sequential fluid flow without external equipment. This review focuses on recent developments on how to control flow rate in paper-based microfluidic devices for automating sequential multi-step assays. The aim of this review is to discuss the limitations of LFTs and potential paper-based microfluidic devices for automated sequential multi-step assays in developing countries; and the existing fluidic control technologies for sequential multi-step assays. In addition, we present future challenges for commercialization of paper-based microfluidic devices to perform automatic multi-step assays.

An integrated fluorescent lateral flow assay for multiplex point-of-care detection of four respiratory viruses

Schistosomiasis is a significant public health concern, especially in Sub-Saharan Africa. Conventional microscopy is the standard diagnostic method in resource-limited settings, but with limitations, such as the need for expert microscopists. An automated digital microscope with artificial intelligence (Schistoscope), offers a potential solution. This field study aimed to validate the diagnostic performance of the Schistoscope for detecting and quantifying Schistosoma haematobium eggs in urine compared to conventional microscopy and to a composite reference standard (CRS) consisting of real-time PCR and the up-converting particle (UCP) lateral flow (LF) test for the detection of schistosome circulating anodic antigen (CAA). Based on a non-inferiority concept, the Schistoscope was evaluated in two parts: study A, consisting of 339 freshly collected urine samples and study B, consisting of 798 fresh urine samples that were also banked as slides for analysis with the Schistoscope. In both studies, the Schistoscope, conventional microscopy, real-time PCR and UCP-LF CAA were performed and samples with all the diagnostic test results were included in the analysis. All diagnostic procedures were performed in a laboratory located in a rural area of Gabon, endemic for S. haematobium. In study A and B, the Schistoscope demonstrated a sensitivity of 83.1% and 96.3% compared to conventional microscopy, and 62.9% and 78.0% compared to the CRS. The sensitivity of conventional microscopy in study A and B compared to the CRS was 61.9% and 75.2%, respectively, comparable to the Schistoscope. The specificity of the Schistoscope in study A (78.8%) was significantly lower than that of conventional microscopy (96.4%) based on the CRS but comparable in study B (90.9% and 98.0%, respectively). Overall, the performance of the Schistoscope was non-inferior to conventional microscopy with a comparable sensitivity, although the specificity varied. The Schistoscope shows promising diagnostic accuracy, particularly for samples with moderate to higher infection intensities as well as for banked sample slides, highlighting the potential for retrospective analysis in resource-limited settings. NCT04505046 ClinicalTrials.gov.

The recombinant, modified leucine-rich repeat protein rhKU_Sej_LRR_2271 has been suggested as a candidate for leptospiral vaccine development since it was predicted to be a transmembrane protein containing leucine-rich repeat motifs and immunogenic epitopes. The immunogenic epitopes showed binding affinities with lower IC50 values than peptides of known antigenic proteins, e.g., LipL32. Moreover, this protein was immunoreactive with hyperimmune sera against several serovars. In this study, we aimed to develop a lateral flow strip test using the rhKU_Sej_LRR_2271 protein for the detection of anti-leptospiral IgG in dogs. The lateral flow assay was performed with 184 dog plasma samples and evaluated with a culture method, 16S ribosomal RNA gene (rss) analysis real-time PCR, and LipL32 ELISA. The culture method failed to detect leptospires in the dog blood samples. Six of nine symptomatic dogs gave positive results with the real-time PCR assay. The lateral flow assay and LipL32 ELISA gave positive results with 59 and 50 dogs, respectively. The sensitivity, specificity, and accuracy of the rhKU_Sej_LRR_2271 lateral flow strip test were 70.00, 82.09, and 78.80%, respectively, when compared with LipL32 ELISA. There was a significant association between the LipL32 ELISA and the rhKU_Sej_LRR_2271 lateral flow assay. The rhKU_Sej_LRR_2271 lateral flow strip test has therefore demonstrated a good potential to detect anti-leptospiral IgG in dogs.

Invasive aspergillosis (IA) remains a potentially lethal disease and requires timely diagnosis and initiation of antifungal therapy. Recently, the IMMY lateral flow assay (LFA), the OLM Diagnostics lateral flow device (LFD), and the Wako turbidimetric β-d-glucan assay have been approved for use as a diagnostic aid. However, their performance in diagnosing IA on serum samples from at-risk patients and the added value to the existing detection of serum galactomannan remain to be investigated. We prospectively collected serum samples from 239 hematology patients and evaluated the diagnostic performance of these 3 assays while using the 2019 EORTC/MSG definitions (study number S59863/S61797, NCT03004092). We identified 5 cases of proven IA, 36 cases of probable IA, and 188 controls. The LFA had the highest negative predictive value (NPV) and sensitivity (0.90 and 0.49, respectively) while galactomannan detection had the highest positive predictive value and specificity (0.93 and 0.99, respectively). Sensitivity was not significantly different between both tests. When used in combination, the highest NPV was seen in patients with a negative LFA and a negative β-d-glucan test. The sensitivity of the LFD was significantly lower than the LFA. After omitting serum galactomannan from the definitions to control for incorporation bias, the sensitivity of the LFA outperformed galactomannan detection (0.41 vs 0.31, P = .046). The LFA is a fast and effective alternative to serum galactomannan detection for the diagnosis of IA and is especially useful for centers with low sample throughputs. The addition of the Wako β-D-glucan assay further improves the diagnostic performance.

Serological and molecular detection of infection with Mycobacterium leprae in Brazilian six banded armadillos (Euphractus sexcinctus)

Sensitive detection of microRNA-21 in cancer cells and human serum with Au@Si nanocomposite and lateral flow assay

The EIAs (enzyme immunoassays) that react with the SARS-CoV-2 RBD protein and Spike protein such as DiaSorin S1/S2 IgG and Abbott II IgG correlate very well with neutralizing antibodies. LFAs (lateral flow assays) are the fastest and most convenient tests usually performed within 10 – 15 minutes. Several LFAs have been commercialized to detect IgG, IgM antibodies. In this short report we characterised the Dynamiker SARS-CoV-2 neutralising antibodies (Nabs) LFA with EIAs (DiaSorin Liaisons SARS-CoV-2 RBD antibodies and Abbotts IgG spike antibodies) in the sera of Covid-19 convalescent patients (40 – 93 days) post onset of symptoms. The EIAs (DiaSorin Liaisons SARS-CoV-2 RBD antibodies and Abbotts IgG spike antibodies) were detected in the sera of all the Covid-19 convalescent patients (100%), while the LFA Dynamiker SARS-Cov-2 Nabs were found in the sera of 86% of Covid-19 convalescent patients.  In addition, the Dynamiker SARS-CoV-2 Nabs lateral flow immunoassay (LFA) had a positive correlation with the DiaSorin Liaison (RBD) and Abbott IgG Quant (Spike) antibody assays.