Biotinylated Phosphorus Dendrimers as Control Line in Nucleic Acid Lateral Flow Tests

Abstract

Lateral flow assays (LFA) are an affordable, easy-touse, qualitative rapid test for clinical diagnosis in nonlaboratory environments and low-resource facilities. The control line of these tests is very important to provide a valid result, confirming that the platform operates correctly. A clear, nondiffused line is desirable. The number of colored nanoparticles that reach the control line in a positive test can be very small, and they should all be trapped efficiently by the molecules adsorbed there. In this work, we proposed the use of robust biotinylated dendrimers of two different generations as signal amplifiers in control lines of LFA, able to react with streptavidin-modified gold nanoparticles. Besides the synthesis and characterization, the analytical performance as control lines will be studied, and their response will be compared with other commercially available biotinylated molecules. Finally, the utility of the dendrimer implemented in a NALF (Nucleic Acid Lateral Flow) strip was also demonstrated for detection of the amplicons obtained by double-tagging PCR (polymerase chain reaction) for the detection of E. coli as a model of foodborne pathogen.

Immunomagnetic Separation Improves the Detection of Mycobacteria by Paper-Based Lateral and Vertical Flow Immunochromatographic Assays

“…The different pads conforming the system were assembled and placed in the cartridge, as shown in Figure 1 . Afterward, 0.5 μL of the anti-digoxigenin antibody (1 mg mL −1 ) and the positive control biotinylated reporter [ 27 ] (1 mg mL −1 ) were deposited on the nitrocellulose membrane and were then dried at RT for 30 min. Furthermore, the streptAv-AuNPs were diluted 5 times in conjugate diluting buffer, embedded in the glass fiber conjugate pads, and then dried for 3 h at RT.…”

“…All of the pads were dried for 3 h at RT. The antibody (1 mg mL −1 ) and the positive control biotinylated reporter (1 mg mL −1 ) [ 27 ] were dispensed on the nitrocellulose membrane and dried at RT for 1h. Finally, the strips were assembled on the adhesive backing card as it is shown in Figure 1 .…”

“…A valid test was considered when the streptAv-AuNPs also reacted with a biotinylated reporter located on the control dot. The reporter is a biotinylated reporter previously described by our research group [ 27 ], although good performance can be achieved by using other biotinylated proteins at higher concentration levels [ 29 ].…”

“…When streptAv-AuNPs/amplicon moved through the nitrocellulose, the specific antibody anti-DIG on the test line reacted with the DIG-tag of the amplicons. The remaining streptAv-AuNPs flowed through the nitrocellulose up to the control line where they were captured by the biotinylated reporter [ 27 ] used as a positive control. After 10 min, the test revealed the lines, and the test was ready for the interpretation of the results.…”

“…Even improved analytical features were achieved by combining double-tagging amplification with immunomagnetic separation (IMS) [ 19 ]. The integration of double-tagged amplicon to RDTs based on immunochromatography was also reported [ 27 , 28 ], showing a comparable sensitivity for the detection of double-tagged amplicons: electrochemical biosensing and lateral flow labeled with Au-NPs [ 28 ] and carbon nanoparticles [ 29 ].…”