Supplemental Figure Legends and Supporting Data.

Verification of QD-MBP-5HIS complex formation and protein:QD ratio. MBP95C-

Cy3 was utilized to monitor of MBP concentration via an absorbance maximum that

would not be masked by QD absorption at ~280 nm. One hundred pmol of 560 QD were

mixed with the molar equivalent amount of MBP95C-Cy3 sufficient to coat each QD

with ~10 MBPs. After mixing, the 560 QD–10MPBCy3 complex was allowed to self

assemble. A control was set up at the same concentration of MBP95C-Cy3, but without

the QDs. The measured absorbance of each solution was equal at the Cy3 absorbance

maximum ~ 553 nm. Each solution was added to an Amicon Centricon spin dialysis unit

(100 kD MWCO) and centrifuged for 20 min at 1000 x g. A wash of borate buffer was

added, followed by a second spin. The dialysate passing through the membrane was

collected and the amount of MBP95C-Cy3 in each sample was determined. More than

95% of the MBP95C-Cy3 in the non-QD control sample passed through the membrane.

In the 560 QD-10 MBP95C-Cy3 sample, less than 3% of the MBP95C-Cy3 present

passed through the membrane. Since the MW of MBP95C-Cy3 is ~ 45 kD and the MW

of the 560 QD-10 MBP95C-Cy3 is estimated to be >500 kD, these results support the

conclusion, together with the data on PL enhancement (Figure 1), that virtually all the

MBP95C-Cy3 present in the sample with the QDs was bound to the QD’s and therefore

could not pass through the membrane. Furthermore, the ratio of MBP95C-Cy3:QD is

~10, since if fewer than 10 MBP95C-Cy3s bound per QD, then a larger percentage of the

MBP95C-Cy3 present in the sample would be expected to pass through the membrane.

For example, if only 9 out of the 10 MBP95C-Cy3s adhered to the QDs, then 10% of the

protein in the experiment would pass through the membrane. It is important to recognize

that on average there are ~10 MBPs around each QD, and that some complexes might

have only 8 to 9 MBPs while others may have 11-12. A Gaussian distribution centered

around ~10 MPBs/QD is a plausible working model.

Supplemental Figure 1B. Effect of imidazole concentration on QD-MBP conjugate formation (fluorescence measured after amylose column)

Wavelength nm (400 nm excitation)450 500 550 600 650

PL

(AU

)

0

1x106

2x106

3x106

4x106

5x106

A.

450 500 550 600 650

PL (A

U)

0

10x106

20x106

30x106

40x106

50x106

MBP-5HIS Ratio of MBP to QD -->

MBP<-- Ratio of MBP to QD

604020

60

PL (A

U)

10x107

40

20

105

1

2

50 mM imidazole

20 mM imidazole10 mM imidazole5 mM imidazole

560 QD + 20 MBP/QD (Post amylose column)

560 QD w/ 20 MBP/QD(Before amylose column)

1 mM imidazole 560 QD w/ 20 MBP/Dot (Washed with 250 mM imidazole while on amylose column)

Supplemental Figure 1A. Comparison of the ability of MBP-5 HIS (penta-histidine) to adhere to QDs compared to MBP (penta-histidine truncation) (fluorescence measured after amylose column)

0

Wavelength nm (400 nm excitation)

20x107

30x107

40x107

50x107

Supplemental Figure 1A. Comparison of MBP-5HIS (penta-histidine) and MBP (penta-

histidine truncated) binding to DHLA-QDs. After the indicated ratios of each protein

were allowed to bind to 560 QDs, the QD-protein complexes were applied to an cross-

linked amylose resin column, washed with buffer and eluted with buffer + 10 mM

maltose. The amount of MBP-QD conjugate specifically eluted was determined by

measuring the PL of the eluted material. QDs without an MBP coating become

irreversibly entrapped in the matrix and will not pass through the amylose column with or

without application of maltose. Note: different x-axis scales are utilized to show the

large difference in concentrations eluted; the scale for MBP-5HIS-QD PL is 10-fold

expanded. (1B) Effect of imidazole concentration on QD-MBP coordination. MBP-

5HIS were allowed to coat 560 QDs at a ratio of 20 proteins per QD in the presence of

the increasing amounts of imidazole as indicated. These conjugated QDs were then

passed over amylose affinity columns, washed and eluted with 10 mM maltose. The

amount of MBP-QD conjugate that eluted was compared by measuring the resultant PL.

In addition, one solution of 560 QD-20 MBP-5HIS was applied to an amylose column

and then washed with 250 mM imidazole while bound. Since imidazole is an analog of

the ring structure found on the histidine residues, this portion of the histidine residue

ostensibly coordinates with the QD surface. Excess imidazole will compete with the

engineered oligohistidine present on MBP-5HIS and eventually outcompete the protein

for binding to the QD surface as the imidazole concentration rises. Indeed, this is the

basis for the oligohistidine-based purification of proteins36. If, as we postulate, MBP-

5HIS-Zn(II) coordination on the QD surface is the basis for QD-MBP self-assembly, then

excess imidazole should adversely affect this coordination, and this is what is observed.

Amylose Resin Assay. 1 mL of crosslinked amylose resin (NEB) was added to a small

chromatographic column and washed 2x with buffer (10 mM NaCl, 10 mM Na-

tetraborate buffer, pH 9.55). The QD-MBP conjugate preparation was applied to the

column followed by washing with 2 mL buffer. QD-MBP complex was eluted with 1 mL

of buffer containing 10 mM maltose. If desired, binding and elution of the QD-MBP

conjugate can be monitored with a hand-held UV light. QDs without an MBP coating

bind irreversibly to the chromatographic matrix and will not pass through the column

with or without application of maltose.

0

1x106

2x106

3x106

1 uM β-CD-Cy3.5Equivalent of 10 MBPCy3/QD10 MBP-Cy3 + 1 uM β-CD-Cy3.5+ 10 mM maltose

B.

PL

(AU

)

0

1x106

3x106

4x106530 QD w/ 10 MBP/QDEquivalent 10 MBPCy3530 QD w/ 10 MBPCy3/QD1 uM β-CD-Cy3.510 MBPCy3 + 1 uM β-CD-Cy3.5Assembled sensor 530QD w/ 10MBPCy3/QD-β-CD-Cy3.5

C.

500 550 600 650 700 750

Supplemental Figure 2. 530 QD w/ 10 MBPD95C-Cy3/QD - β-CD-Cy3.5 controls

0

500x103

1x106

2x106

530 QD530 QD w/ 10 MBP/QD+ equivalent of 10 free Cy3 dye/QD

A.

Wavelength nm

Wavelength nm

500 550 600 650 700 7500

200x103

400x103

600x103

β-CD-Cy3.5

530 QD

MBP-Cy3E.

Supplemental Figure 2. 530 QD w/ 10 MB D95C-Cy3/QD - β-CD-Cy3.5 controls

0 Maltose

10 mM maltose

500 nM maltose

1 µM maltose

5 µM maltose

PL

(AU

)

0

1x106

2x106

530 QD w/ 10 MBP/Dot

530 QD

1 µM β-CD-Cy3.5

530 QD w/ 10 MBP/QD+1 µM β-CD-Cy3.5+ 5 mM maltose

D.

500 550 600 650 7000

500x103

1x106

2x106

2x106 530 QD

MBP-Cy3

Wavelength nm

F.

Time (ns)0 2 4 6 8 10

Nor

mal

ized

inte

nsity

(AU

)

0.0

0.2

0.4

0.6

0.8

1.010MBPCy3 τ = 1.4 +/- 0.1 ns

530QD w/ 10MBPCy3/QD τ = 2.2 ns +/- 0.0 ns

530QD w/ 10MBPCy3/QD + β-CD-Cy35τ = 1.9 +/- 0.1 ns

G.

PL

(AU

)

Supplemental Figure 2. In order to investigate the FRET process and to determine the

direct PL contribution from each individual nanosensor assembly component, a series of

controls was carried out. (A) 530 QD-10MBP-Cy3-β-CD-Cy3.5 controls. Excitation

was at 450 nm (approximate Cy3 absorption minimum). (A) Emission spectra from 530

QDs, 530 QDs conjugated with 10 MBP/QD, and the equivalent concentration of 10 free

Cy3 dye/QD. (B) MBP-Cy3-β-CD-Cy3.5 controls. Emission spectra from the equivalent

amount of 10 MBP monolabeled with Cy3 (molar amount added to 30 pmol of QD for

10MBP/QD), 1 µM β-CD-Cy3.5, the 10 MBP-Cy3 bound to 1 µM β-CD-Cy3.5, and 10

MBP-Cy3 bound to 1 µM β-CD-Cy3.5 after the addition of 5 mM maltose. Note the

almost negligible change in MBP-Cy3 to β-CD-Cy3.5 PL ratio due to excitation at the

Cy3 absorption minimum. (C) 530 QD-MBP-Cy3-β-CD-Cy3.5 sensor assembly.

Emission spectra from 530 QDs conjugated with 10 MBP/QD, the equivalent amount of

10 MBP-Cy3, 530 QDs conjugated with 10 MBP-Cy3/QD, 1 µM β-CD-Cy3.5, 10 MBP-

Cy3 bound to 1 µM β-CD-Cy3.5, and the final assembled sensor, 530QD-10MBP-Cy3-

β-CD-Cy3.5. Note the dramatic loss in QD PL when conjugated with 10 MBP-Cy3 and

the systematic increase in β-CD-Cy3.5 PL. (D) One-step “direct” 530QD-10MBP-β-CD-

Cy3.5 assembly. 530 QDs were conjugated with 10 unlabeled MBP/QD and allowed to

bind with 1 µM β-CD-Cy3.5. The resulting assembly was exposed to 5 mM maltose.

(The arrow points to 1 µM β-CD-Cy3.5 PL and shows the emission of that moiety

measured alone overlapping the sensor + maltose spectrum). Note the negligible

changes and similarity between final sensor + maltose and free 1 µM β-CD-Cy3.5 PL. A

control experiment where 530 QDs conjugated with 10 unlabeled MBP/QD was exposed

to 5 mM maltose and then 1 µM β-CD-Cy3.5 yielded essentially the same result (Data

not shown). (E) Titration of the 530 QD-MBP-Cy3-β-CD-Cy3.5 sensor assembly with

maltose after the direct excitation contributions of MBP-Cy3 and β-CD-Cy3.5 are

subtracted. This leaves only the FRET portion of the sensor assembly titration data. (F)

Data from Figure 4C showing 530 QD and MPBCy3 data deconvoluted with direct

excitation of MBP-Cy3 corrected. (G) Fluorescence lifetime decay curves and τ values

of the Cy3 dye from the 10MBPCy3, 530 QD-10MBP-Cy3 and 530 QD-10MBP-Cy3-β-

CD-Cy3.5 sensor assemblies.

Supplemental Figure 3. β-CD-dye derivative structures25. (A) β-CD-QSY9. (B) β-CD-

Cy3.5. Not to scale. Molecular weights were verified by mass spectral analysis.

Supplemental Table I. Test of QD–MBP sensor specificity using the 560QD-10MBP-

β-CD-QSY9 assembly. The QD-sensor was assembled as described in Methods and

diluted into 3 mL borate buffer in a cuvette for assaying. Two concentrations of each

indicated sugar were added to the cuvette, 100 µM and 1 mM. The ability of each of the

sugars to displace the β-CD-QSY9 from the MBP binding pocket was monitored,

providing a test of MBP recognition specificity in this sensor configuration. The change

(∆) in PL, if any, upon sugar addition was monitored at the QD emission maximum. Any

sugar induced change in PL is reported in the table as the % change in PL compared to

the sensor assembly PL prior to sugar addition. The type and saccharide linkage of each

sugar tested is also described on the table. Soluble MBP (i.e., uncomplexed protein)

specifically binds sugars that contain an α-1,4 glucosidic linkage31. As demonstrated in

the table, only those sugars with this type of saccharide linkage are able to displace β-

CD-QSY9 and increase QD PL. These assays demonstrate that MBP coordinated to QDs

retains its sugar binding specificity.

Supplemental Table II. Estimated energy transfer efficiencies for indicated donor-

acceptor pairs in the sensor assemblies. Efficiencies were estimated in two ways. First,

the direct donor PL loss as described in Methods26 was used in calculations of E.

However, since this does not account for quenching of the donor by other non-FRET

radiative and non-radiative decay processes, FRET efficiencies were also calculated from

direct-excitation-corrected acceptor PL data where possible. Where the acceptor was a

QSY9 dark quencher, an average FRET efficiency based on the other derived data was

used for estimation purposes. However, since the J(λ) and 560 QD-QY is better here,

overall FRET efficiency will probably be higher than estimated for the 560QD-QSY9

pair.

Estimated FRET Relay Efficiency34

Efficiency: D

ADc F

FE =

Where FD is the relative fluorescence of the donor in the absence of acceptor and FAD is

the fluorescence of the acceptor in the presence of donor and corrected for the direct

excitation of the acceptor and any other non-FRET contribution from the donor as

described in Methods.

The FRET process in the 530 QD-10 MBPCy3-β-CD-Cy3.5 assembly can be described

as:

E13

530QD 10 MBPCy3 β-CD-Cy3.5 (1) E12 (2) E23 (3)

Where Eβ-CD-Cy3.5 = E12 * E23 + E13

= 71% * 20% + 6%

= 20%

Eβ-CD-Cy3.5 is the estimated energy β-CD-Cy3.5 receives (FRET only) when bound in the

saccharide pocket of the 530 QD-10 MBPCy3 assembly. The values used are derived

from data such as that in supplementary Figures 2A-C and are all corrected for direct

excitation components.

Supplementary Table I. Quantum Dot - MBP Sensor Assembly Specificity (560QD-10MBP-β-CD-QSY9) Sugar D-Arabinose β-Cyclodextrin D-Galactose D-Glucose D-Mannitol Maltose Sucrose Starch Type1 Mono Oligo (7-monomers) Mono Mono Mono Di Di Oligo

Oligosaccharide Linkage

NA α-1,4 glucosidic

NA NA NA α-1,4 glucosidic

α-1, β-2 glucosidic

α-1,4 glucosidic

∆ PL2 100 uM < 1% 47% < 1% < 1% < 1% 96% < 1% 55%

∆ PL2 1 mM < 1% 54% < 1% < 1% 1.5% 104% 1.6% 58% 1Mono, Di and Oligo – saccharide 2∆ PL = Percentage increase in PL intensity with respect to the initial value of the nanosensor, upon addition of the indicated amount of sugar NA-Not applicable Supplementary Table II. Estimated Energy Transfer Efficiency of Sensor Assemblies

Donor Acceptor Estimated Efficiencies from Donor ∆PL

Estimated Efficiencies Corrected from Acceptor ∆PL

Comments

530 QD + 10 MBP/QD 1 uM β-CD-Cy3.5 ~ 13% ~ 6% MBP adhered to QD surface 530 QD 10 MBP-Cy3/QD ~ 94% ~ 71% MBP-Cy3 adhered to QD surface

0.1 uM MBP-Cy3 1 uM β-CD-Cy3.5 ~12% ~20%* No QD present 530 QD + 10 MBP-Cy3/QD 1 uM β-CD-Cy3.5 ~ 25% ~ 20% MBP-Cy3 adhered to QD surface

560 QD + 10 MBP/QD 1 uM QSY9 free dye ~ 7.5% < 6 % MBP adhered to QD surface 560 QD + 10 MBP/QD 1 uM β-CD-QSY9 ~ 47% ~ 35% MBP adhered to QD surface

λ Excitation = 450 nm The amount of QD used was 30 pmol. per experiment26. *The low value is due to exciting close to a Cy3 absorption minima