temperate phage λ  (ATCC)

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: DNA Hanger: Surface‐Minimized Single‐Molecule Immunoassay Platform

doi: 10.1002/smll.202409933

Figure Lengend Snippet: DNA Hanger. a) The 3D‐etched quartz surface features narrow ridges (≈1 µm wide) with a height of 4 µm, which enables the formation of a thin excitation light sheet near the ridges under prism‐based TIRF illumination. [ ¹³ ] A 3′digoxigenin oligonucleotides were ligated to 12 nt cohesive ends of λ‐phage DNA. The λ‐phage DNA molecules are suspended between two adjacent ridges via specific binding to an anti‐digoxigenin antibody, effectively forming a surface‐suspended bridge structure. b) Biotin groups were introduced onto nucleic acid bases using a photo‐coupling reagent (photobiotin), which covalently attaches biotin upon 405 nm irradiation. In an alternative approach, biotin was site‐randomly incorporated into double‐stranded DNA using a pSoralen–biotin conjugate, a photoactivatable reagent that intercalates into DNA and covalently crosslinks to thymine bases upon UV exposure (365 nm). c) Representative images showing Cy5‐streptavidin binding to biotin‐coated λ‐phage DNA (upper left). The bottom right panels display 5′ biotin‐ssDNA‐Cy5 binding to streptavidin ‐coated λ‐phage at various concentrations of the ssDNA‐Cy5. d) A plot showing fluorescence intensity of Cy5 as a function of 5′biotin‐ssDNA‐Cy5 concentrations using DNA Hanger. Each concentration was repeated in two independent experiments ( N = 2), with the number of field of view (FOVs) as follows: n = 4, 7, 8, 13, 10, 7 and 9 for 6.4 p m , 3.2 × 10 p m , 1.6 × 10 2 p m , 8.0 × 10 2 p m , 4.0 × 10 3 p m , 2.0 × 10 4 p m , and 1.0 × 10 5 p m , respectively.

Article Snippet: The DNA substrate was prepared using bacteriophage λ‐phage DNA (48.5 kbp, New England Biolabs) through ligation with 14 nt‐length 3′ digoxygenin‐labeled oligonucleotides, using T4 ligase (NEB) at room temperature overnight (Table , Supporting Information).

Techniques: Binding Assay, Irradiation, Fluorescence, Concentration Assay

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: DNA Hanger: Surface‐Minimized Single‐Molecule Immunoassay Platform

doi: 10.1002/smll.202409933

Figure Lengend Snippet: Capture of PABP bound to RNA. a) Schematic (top) and corresponding representative fluorescence images (bottom) showing the stepwise assembly of the assay: Cy5‐DNA/RNA duplex containing a poly(A) 114 RNA tail (red) tethered to the λ‐phage DNA bridge via a streptavidin–biotin interaction (left), binding of mNG‐PABP (green) to the duplex (middle), and subsequent binding of AF594‐anti‐PABP antibody (pink) to mNG‐PABP (right). Magnified views of the white boxed regions are shown on the far right. White arrows indicate colocalized spots exhibiting all three fluorescence signals. b) Non‐colocalization ratios between mNG‐PABP and AF594‐anti‐PABP antibody in the flat PEG–biotin‐coated quartz surface ( n = 41, N = 3 for both mNG‐PABP and AF594‐anti‐PABP antibody) and the DNA Hanger platform ( n = 18, N = 3 for mNG‐PABP, n = 17, N = 3 for AF594‐anti‐PABP antibody). Yellow and green diamonds represent the normalized non‐colocalization ratios for mNG‐PABP and AF594‐anti‐PABP antibodies, respectively (see the for the mathematical definition). Error bars represent standard deviations (S.D.).

Article Snippet: The DNA substrate was prepared using bacteriophage λ‐phage DNA (48.5 kbp, New England Biolabs) through ligation with 14 nt‐length 3′ digoxygenin‐labeled oligonucleotides, using T4 ligase (NEB) at room temperature overnight (Table , Supporting Information).

Techniques: Fluorescence, Binding Assay

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: DNA Hanger: Surface‐Minimized Single‐Molecule Immunoassay Platform

doi: 10.1002/smll.202409933

Figure Lengend Snippet: Single‐molecule FLISA of TNF‐α in buffer. a) Schematic of TNF‐α detection using DNA Hanger. AF647‐cAb is immobilized on streptavidin‐coated λ‐phage DNA through the biotin‐streptavidin interaction. TNF‐α is captured by the cAb and subsequently recognized by dAb. b) Quantification of nonspecific binding of dAb (10784 λ‐phage DNA molecules, n = 30, and N = 3) and TNF‐α (6894 DNA, n = 30, and N = 3) to DNA Hanger or to cAb (9954 DNA, n = 30, and N = 3). Each diamond indicates the average of the number of dAb per DNA in a field of view. Statistical analysis was performed using one‐way ANOVA followed by Tukey post hoc test ( ** p < 0.01). “ns” denotes no statistically significant difference ( p ≥ 0.05). c) Representative fluorescence images showing cAb on λ‐phage DNA and dAb bound to TNF‐α at a concentration range (0 – 1000 p m ). d) Quantification of dAb binding per λ‐phage DNA at varying TNF‐α concentrations (mean ± S.D.): 0.07 ± 0.04 at 0 p m (9951 DNA, n = 30, and N = 3), 0.31 ± 0.11 at 3 p m (3465 DNA, n = 17, and N = 3), 1.31 ± 0.47 at 10 p m (3714 DNA, n = 21, and N = 4), 3.41 ± 0.45 at 30 p m (1769 DNA, n = 18, and N = 3), 9.92 ± 1.04 at 100 p m (473 DNA, n = 9, and N = 3), 16.3 ± 1.93 at 300 p m (518 DNA, n = 6, and N = 2), and 17.5 ± 1.60 at 1000 p m (736 DNA, n = 6, and N = 3).

Article Snippet: The DNA substrate was prepared using bacteriophage λ‐phage DNA (48.5 kbp, New England Biolabs) through ligation with 14 nt‐length 3′ digoxygenin‐labeled oligonucleotides, using T4 ligase (NEB) at room temperature overnight (Table , Supporting Information).

Techniques: Fluorophore-linked Immunoabsorbent Assay, Binding Assay, Fluorescence, Concentration Assay