Pan-Chyr Yang
*
Supplementary figures and text:
Supplementary Figure 1: Flowchart for cell-based SELEX.
Supplementary Figure 2: A dendrogram analysis of 96 oligonucleotide sequences after 8 rounds of cell-based SELEX.
Supplementary Figure 3: Result of fluorescence microscopy of SA17 and
SA61 against different bacteria.
Supplementary Figure 4: Dissociation constant (K d
) and predicted structures of SA17 and SA61.
Supplementary Figure 5: Binding capacity of SA17 and SA61 on S. aureus cells.
Supplementary Figure 6. Binding capacity of SA17 and SA61 on 60 nm
GNPs.
Supplementary Figure 7: Characterization the light scattering signal of GNPs.
Supplementary Figure 8: Single bacteria detection by bead-based amplification.
Supplementary Table 1: The interaction of SA aptamers (SA17 and SA61) with 21 bacteria strains
Supplementary Table 2: Aptamer sequences in round 8 pool.

Supplementary Figure 1.
Flowchart for cell-based SELEX. The selection process is based on hybridization of S. aureus with a library of DNA aptamers in solution followed by counter-selection using S. epidermidis .

Supplementary Figure 2.
A dendrogram analysis of 96 oligonucleotide sequences after eight rounds of cell-based SELEX.

Supplementary Figure 3.
Fluorescence microscopic detection of SA17 and SA61 binding to different bacteria.
Biotin-labeled SA17 or SA61 (500 nM) were incubated with S. aureus cells at
4°C for 30 minutes and stained with streptavidin-PE. Paired sets of images for each of the tested bacterial strains incubated with SA17 (left pair) and SA61 (right pair) are shown. Left image in each pair: bright field; right image in each pair: fluorescence microscopy.

Supplementary Figure 4. K d s and predicted structures of SA17 and SA61.
(A) K d measured by a total binding assay based on quantification of bound SA17 (left) and
SA61 (right) aptamers by qPCR. Different concentrations of aptamers were incubated with equal numbers of S. aureus cells; bound aptamers were eluted and quantified by qPCR. The calculated K d s of SA17 and SA61 for S. aureus binding were 35 and 129 nM, respectively. (B) Secondary structures predicted with mfold software for SA17
(left) and SA61 (right). ∆G values represent the stability of the structures. (C) K d s for
SA17-GNPs (left) and SA61-GNPs (right) were determined by quantification of bound aptamer-GNPs. Different concentrations of aptamer-GNPs were incubated with equal numbers of S. aureus cells, and bound aptamer-GNPs were eluted and quantified. The
K d s of SA17-GNPs and SA61-GNPs for S. aureus were 3.03 and 9.9 nM, respectively.

Supplementary Figure 5. Binding capacity of S. aureus cells for SA17 and SA61 .
(A) SA17 (250 nM) was incubated with samples containing 10, 100, 1000, and 10,000
S. aureus cells; a no-cell control was included as a background signal for calculation of

C
T
. After removing unbound aptamers, bound aptamers were eluted by heating and quantified by qPCR. The results are shown in the upper panel. The number of bound SA17 molecules per S. aureus cell is calculated from the

C
T
by reference to the standard curve shown in the lower panel. (B) The results for SA61 aptamers

obtained as in A.

Supplementary Figure 6. Binding capacity of 60 nm GNPs for SA17 and SA61 . (A)
Thio-adaptor sequences (5

M) were conjugated onto 60-nm GNPs, and adaptor-GNPs were further incubated with 5

M SA17 aptamer. After washing away the unbound aptamers, SA17-GNPs were quantified by measuring OD
550
. Different numbers of SA17-GNPs (10, 100, 1000, and 10,000) were analyzed by qPCR to calculate the amount of bound aptamer; buffer alone was used as a background control.

(B) Binding capacity of GNPs for SA61 determined as in A.
Supplementary Figure 7. Characterization of the light-scattering signal of GNPs. a ,
Light-scattering signals from serially diluted samples of 60-nm GNPs. The highest concentration is 3

10 5 particles/
 l. The control is buffer alone. b , Light-scattering intensity of different sizes (15, 30, 60, and 100 nm) and concentrations of GNPs. The scattering results indicated that the signal is increased in proportion to particle size and concentration. The detection limit for 100, 60, 30, and 15 nm GNPs were 63

21, 508

176, 7.8

10 4 , and 5

10 6 particles/
 l, respectively. Open circle: 100 nm GNPs; open square: 60 nm GNPs; triangle: 30 nm GNPs; inverted triangle: 15 nm GNPs. c , The light-scattering data fit to a nonlinear equation showing that signal intensity increases with the sixth power of the particle radius, consistent with previous findings 27 .

Supplementary Figure 8. Detection of a single bacterium by bead-based amplification . Bacterial suspensions containing 10 bacterial cells, estimated based on
OD
600
values, were divided equally into 30 samples. Biotin-SA61-GNPs and SA17-M270 beads were added to each sample followed by application of the bead-based amplification protocol. The signal intensity of each sample was measured as above. Four independent

assays were performed, with stars marking positive wells containing bacteria. The three gray bars at right show the signal for no-bacteria controls, and the red dashed line indicates the highest signal intensity in the three controls. The signal intensities of wells above the red dashed line (positive wells) are marked with asterisks. In four independent assays, 12, 19, 8, and 18 positive wells were obtained.
Binomial Nomenclature
Bacillus subtilis
Citrobacter freundii
Escherichia coli
Klebsiella pneumoniae
Listeria monocytogenes
Moraxella catarrhalis
Pseudomonas aeruginosa
Salmonella enterica
Shigella boydii
Shigella flexneri
Staphylococcus aureus Strain 0
Staphylococcus aureus Strain 1
Staphylococcus aureus Strain 2
Staphylococcus aureus Strain 3
Staphylococcus aureus Strain 4
Staphylococcus aureus Strain 5
ATCC Number
21336
8090
43896
13883
19112
25238
27853
13314
8700
29903
6538DR
6538P
12600
25923
29213
6538
SA17
—
—
—
—
—
—
—
—
—
+
+
+
+
—
+
+
SA61
—
—
+/-
—
—
—
—
—
—
+
+
+
+
—
+
+

Staphylococcus epidermidis
Staphylococcus haemolyticus
Staphylococcus saprophyticus
Streptococcus bovis
155
29970
15305
43077
—
—
—
—
+/-
—
—
—
Streptococcus pneumoniae 6301
— —
Supplementary Table 1. The interactions of SA aptamers (SA17 and SA61) with 21 bacterial strains, including six S. aureus strains and 15 bacteria from other genera or species. Binding assays were performed using IFA/fluorescence microscopy. Scoring of binding-signal ratios of aptamers to S. aureus relative to negative controls in IFAs: “-“, ratio < 1.5-fold; “+/-”, ratio = 2–3-fold; “+”, ratio > 3-fold.

SA-1
TCCCTACGGCGCTAACCCACTCCCCTCCACCGCTCCGACTCCGTCCGCCACCGTGCTACAAC
SA-23
SA-24
SA-25
SA-26
SA-27
SA-28
SA-29
SA-30
SA-31
SA-32
SA-33
SA-9
SA-10
SA-11
SA-12
SA-13
SA-14
SA-15
SA-16
SA-17
SA-18
SA-19
SA-20
SA-21
SA-22
SA-2
SA-3
SA-4
SA-5
SA-6
SA-7
SA-8
TCCCTACGGCGCTAACCTCACACCGACCTGCTTCCCCCCCCCGGCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCCCCAGTCCGTCCTCCCAGCCTCACACCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCATCACCGCACCTCCCACCGACTCCCCTGCCACCGTGCTACAAC
TCCCTACGGCGCTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCACCGCTCTCGCCCAGCTCCTCTCCTGCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCTCCTCCCACCTCGCCCAGTCCGCCTACGCCACCGTGCTACAAC
TCCCTACGGCGCTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
TCCCTACGTGATTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCAGCCACGTCCCGTCCACCCCGCCACCTCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCTGCTCCCCCCACCGTGTCCTCGCCTACGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCACCCCCCCGGACCCGCTCTCCTGCCACTCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCTCACATCACTCCCCTCACCGCTACCCACCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCTCCTCGTCACCCTGCGCTCCCACCTCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCCCCAGTCCGTCCTCCCAGCCTCACACCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCCCGCTCCCACGCTCTGCCCTCCTACCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCACTCCTCTCCCGCAGCGCTTCCACCTCCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCTGCTCCTCCCCGGTCCTCCAGCCTCCACCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCACTCCAGCATCCACCCTCCAGCCAACCCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCAGTCCCGTCTCCCACCACGCCCCAGCTGCCACCGTGGTAAAAA
TCCCTACGGCGCTAACCCCTCCCCAGCCATCCTCCGCCACTCCACCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCACCACCACTCCTCTCACCACGCACTCCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCTCACCAGTCCCCCGTCCCTCTCCCGTCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCACACTCCCCGTCACCGCTCCACCGCCAGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCCAACCGTCAGCTCACCCCGTCCTCCCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCTCCACCGAACCTCCCACGCTCCCCGCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCCTGCCCACTCCACACCGTCACCACACCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCTCTCACCGGTCGTCCTCCCCACCTCCAGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCAGTCAACCTCCCCGTCCTCCCGCCAACGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCACTGTCCCCCGTCCCTCCGAGCCTCTCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCTCCCGCTCACTCACCCGTCCCGCCTACGCCACCGTGCTACAAC

SA-62
SA-63
SA-64
SA-65
SA-66
SA-67
SA-68
SA-70
SA-71
SA-72
SA-73
SA-47
SA-48
SA-49
SA-50
SA-51
SA-52
SA-54
SA-55
SA-56
SA-57
SA-58
SA-59
SA-60
SA-61
SA-34
SA-36
SA-37
SA-38
SA-39
SA-40
SA-41
SA-42
SA-45
SA-46
TCCCTACGGCGCTAACACGACAACCGCTACCCCGTCCAGCTCCCCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCTCCACCGGCTCCCTCGCTACCCCACCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCACTCCTCCCCGCCTGGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCCACTCCTCCATCCCGTCGCCCTCCATCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCTCCCCCCAGCTCCTCTCCACCTCGCCTGGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCTCCTCACCCCGCGTCCTCCCACGTCTCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCTCCTCCCACCACAGACCGACTCCCCTCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCTCCTCCCACCACCAGCCGCTCAACTCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCTCCTACCGTCCACCCCCACAGCTCCTCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCCTCCGTCACCGCTCCCACCACCGTCCGGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCTCCACACCCGCTGCCCTCCGTCCTCCCTGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCTCCTCCAACCGTCCCACCCTGCCACTCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCACCTCCGCTACCCTGCCAGCCCTCCCCCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCTCCCGTCCAGTCGTCCTCGCCCCCAACGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCACCATCCCGGCCAAGCTCCACAAGTCCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCACTCCCCAGAAGCCTCCACCGAACGCCAGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCTCCTCACCCCGCGTCCTCCCACGTCTCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCGGTTGGGGTGGTGGGGGAGGGCCAGAGGAGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCTCCCCGCAACACGCACTCCCTGTCTCCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCCAACGCTCTCCCTGCCCCGCGACGCGAGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCTCCCCGTCACCGCTCACCACCGTCCTCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCCCAGCTCTCCCTCCGATCCCAGTCACCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCTCCCAACCGCTCCACCCTGCCTCCGCCTCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCCCTCGCTCCCGCACACCACCACCGACCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCTCTCCGTCCCCCTCCAGCCAACCTCCGGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCACCAGCTCTCCCGTCTCCCCCCGCCTCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCACCTCCACCCGTCCATCCCCGAACCCTCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
TCCCTACCACAGTGGCGGAGGGGTGAGACTGGTTGCGAGGACCGGGGTTAGCGCCGTAGGGA
TCCCTACGGCGCTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCCCAGAAACCTCCGCCTCACCGCCACCAGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCTCCCCGTCACCGCTCACCACCGTCCTCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCTCCTCCTCCACCGCGAACCGTCCCAGATGCCACCGTGCTACAAC
TCCCTACGGCGCTAACTCCCGGAACCCCCATCCCGCTCCACCGCCCGCCACCGTGCTACAAC

SA-84
SA-85
SA-86
SA-87
SA-88
SA-89
SA-90
SA-91
SA-92
SA-93
SA-94
SA-95
SA-96
SA-74
SA-75
SA-76
SA-77
SA-78
SA-79
SA-80
SA-81
SA-82
SA-83
TCCCTACGGCGCTAACCCGTCCACTCCCCGCTACCCAGGTCCTCCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCTCACACAGGCTCTCCTCCGCGACCACCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCTCCACACCGCTCCTCCCAACCGCCTACGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCATCGATGCACCCTCGCCTCTCCTAACCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCACCTCCACCCCAGCCCAGACGTCGCCTCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCACCCTCCTCACCACGTCCCGCCACCACCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCCCCAGTCCGTCCTCCCAGCCTCACACCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCAGCTCCTCTCCCAGCCAAGCCACCCGTGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCCTCACAACCTCACAAGACCGCCCTCCTGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCACCGCTCCTCCACCTCCAGCCGACGCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCACTCGTCTCCCCCCATCACCGCTACCCCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCACCTCCTCCGCACCTCTCCTACGCCTCCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCGCATCCCTCCGCCCTCCTACCCTCCCCGGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCCAGGCTCACCCACCACCGCACCTCTCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCCACGCTCCCAACCTCCCGTCCTCCCCTGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCATCCACGCTCCACCCCCACGTCTCTCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCACGACTCCTCTGCCAACCCGTCCTGACGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCTCACACCCACCGACCGCTCTCCGCCTCCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACGATCGAGACCGTCCAGAGGTTCGAGTGGTAGCCACCGTGCTACAAC
TCCCTACGGCGCTAACTCCACCCGCATCCCCCTCGTCCTACCCTCCGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCCTCCTCCCAGCCTCTCCGCCCTCGAAACGCCACCGTGCTACAAC
TCCCTACGGCGCTAACCCTCCAACCCGTCCACTCCACAACACCCCGGCCACCGTGCTACAAC
Supplementary Table 2.
Aptamer sequences in the round 8 pool.