Supporting Information Single-Digit Pathogen and ... AWS
Supporting Information Single-Digit Pathogen and Attomolar Detection with the Naked Eye using Liposome-Amplified Plasmonic Immunoassay
Minh-Phuong Ngoc Bui, Snober Ahmed, and Abdennour Abbas*
Department of Bioproducts and Biosystems Engineering, University of Minnesota Twin Cities, MN, 55108-6005, USA
*Corresponding author Dr. Abdennour Abbas, Department of Bioproducts and Biosystems Engineering, University of Minnesota Twin Cities, MN, 55108-6005, USA Email: [email protected] Phone: +1 (612) 624-4292
1
Supporting Discussions Calculation of the number of liposomes in solution The number of lipid molecules (Ntot) in a unilamellar liposome (100 nm) was calculated according to equation (1):
=
4 + 4 − ℎ 2 2
(1)
where d is the hydrodynamic diameter obtained by light scattering measurement, h is the bilayer thickness, and αL is the average head group surface area per lipid. The lipid bilayer thickness was assumed to be 5 nm and average αL value for phosphatidylcholine, phosphoethanolamine and cholesterol were 0.65 ± 0.01 nm2, 0.52 ± 0.01 nm2, and 0.41 nm2, respectively.1 Using those values and the molar fraction of each component, the αL value obtained for our produced liposomes was 0.6 nm2/lipid. The Ntot was calculated to be 94771 lipid molecules per liposome of 100 nm. The number of liposomes per milliliter (Nlipo) can be derived from the lipid concentration with Avogadro’s number as shown in equation (2) =
× (2) × 1000
Where NA is Avogadro’s number, and Mlipid is the molar concentration of lipid. The number of liposomes was calculated based on the number of lipid molecules in a liposome and the lipid concentration used to fabricate the liposome solution. Briefly, the liposomes were analyzed with a dynamic light scattering particle analyzer (NanoFlex, Microtrac, USA). A mean diameter size of 81.9 ± 20.5 nm was determined. The calculation showed that the average encapsulated volume of a liposome is 3.8 x 106 nm3 (with an assumption that a lipid bilayer thickness is 5 nm for phosphatidylcholine and phosphoethanolamine). Given that the total concentration of lipid used to fabricate the liposomes is 84.6 µM, the number of liposomes Nlipo was calculated to be 5.38 x 1012 liposomes per mL. During the fabrication process, the liposome solution lost during extrusion and dialysis step was estimated to be around 10 ± 3% and 42.8 ± 2%, respectively. The data were evaluated preparing similar liposomes with fluorescent phosphatidylcholine lipids and analyzed with UV-vis spectrometry. Thus, the number of liposome recovery after preparation is calculated to be 2.77 x 1012 liposomes per mL.
2
Calculation of AuNPs aggregation degree in LAPIA assay Given that the plasmonic peak of aggregated AuNPs shifts to higher wavelengths (from 600 – 700 nm) depending on the aggregation degree, the most accurate way to analyze the data is to use UV-vis absorption spectrometry since it covers all the wavelength range. The microplate reader can only be used to measure the optical density at a single wavelength (655 nm). Hence, the results obtained with the microplate reader are only accurate when the maximum peak of aggregated AuNPs is at around 655 nm, which is the usual wavelength at which the optical density is obtained. At this wavelength, the results of the optical density obtained on the microplate reader are comparable to the absorption data obtained with UV-vis spectrometry. The AuNPs aggregation percent was calculated based on the peak area ratio of single AuNPs (520 nm) and aggregated AuNPs (from 600 – 700 nm) using the peak analyzer function in Origin software (Origin Pro v9.1, Originlab Corp., USA). The peak surface area ratio is used here to quantify the assembly degree of AuNPs. The ratio is normalized to express the assembly rate as a percentage change with the total assembly corresponding to 100%.2
Table S1. Characteristics of the produced liposomes and AuNPs, as measured by dynamic light scattering and TEM.
Sample
Particle size (nm)
Zeta potential (mV)
pH
AuNPs
15 ± 2
-68.1
5.9
Liposome-cysteine-PEG-biotin (100 nm)
82 ± 20
-36
4.0
Liposome-cysteine-PEG-biotin (200 nm)
242 ± 10
-30
3.9
Liposome-cysteine-PEG-biotin (400 nm)
355 ± 18
-26
4.1
3
Figure S1. Effect of pH on the ionization degree (%) of L-cysteine in water, based on its acidic (pK1 = 1.92) and basic (pK2 = 8.37) pKa values.
4
Figure S2. TEM image of PBS buffer with Tween 20 (0.05%) used to hydrolyze Cys-liposomes in LAPIA assay. The image does not show any vesicular structures, confirming the identity of the hydrolyzed liposomes in the TEM images of Figure 2c.
5
Figure S3. (a) Optical photograph of LAPIA test showing the effect of different concentrations of Cys-liposome on the aggregation of AuNPs. Liposome hydrolysis was caused by adding 20 µL PBST solution. (b) Correlation plot showing the aggregation degree as a function of Cysliposome concentration. The number of Cys-liposome was calculated based on their hydrodynamic diameter size and lipid concentration as mentioned above. AuNPs solution, adjusted to pH 7.25 using NaOH 0.5 M and 100 µL was added to each well followed by 40 µL of biotinylated Cys-liposome.
6
Figure S4. Schematic of the Liposome-Enhanced Nanoparticle Aggregation Immunoassay (LAPIA) for rabbit IgG detection. Difference concentrations of rabbit IgG were used as antigen. The reaction proceeds in different steps: (a) Sandwich capture of IgG target by capture anti-IgG immobilized on the plate and biotinylated anti-rabbit IgG antibody. (b) After incubation and washing, streptavidin is added to interact to biotinylated anti-IgG. (c) Biotin-conjugated Liposomes containing cysteine are added to the immunocomplex followed by AuNPs solution. (d) The addition of PBST-1X causes the breakdown of the liposomes and the release of cysteine, leading to immediate aggregation of gold nanoparticles and color shift from red to dark-blue (e).
7
Figure S5. Optical images of LAPIA plate showing the cross-reactivity in the detection of foodborne bacteria. Three microplates were coated with monoclonal anti-E.coli (A), antiSalmonella (B) and anti-Listeria (C). After the washing step, different concentrations of foodborne bacteria suspensions diluted in PBS-BSA 1% buffer solution, were added to the microplates. Specifically, Salmonella typhimurium, Listeria monocytogenes and E.coli O157:H7, were respectively incubated in microplates A, B and C. The detection was performed as previously described. The results clearly show high selectivity and absence of cross-reactivity on in the LAPIA test (the “blue spot” in (A) showed the false positive result in the LAPIA test).
8
Figure
S6.
Chemical
structure
of
L-cysteine,
L-α-phosphatidylcholine
(PC),
phosphoethanolamine-conjugated biotin used in liposome production, including 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000 labeled "PEG2000biotin").
References (1) (2)
Leekumjorn, S.; Sum, A. K. Biophys. J. 2006, 90, 3951-3965. Abbas, A.; Kattumenu, R.; Tian, L.; Singamaneni, S. Langmuir 2013, 29, 56-64.
9
Minh-Phuong Ngoc Bui, Snober Ahmed, and Abdennour Abbas*
Department of Bioproducts and Biosystems Engineering, University of Minnesota Twin Cities, MN, 55108-6005, USA
*Corresponding author Dr. Abdennour Abbas, Department of Bioproducts and Biosystems Engineering, University of Minnesota Twin Cities, MN, 55108-6005, USA Email: [email protected] Phone: +1 (612) 624-4292
1
Supporting Discussions Calculation of the number of liposomes in solution The number of lipid molecules (Ntot) in a unilamellar liposome (100 nm) was calculated according to equation (1):
=
4 + 4 − ℎ 2 2
(1)
where d is the hydrodynamic diameter obtained by light scattering measurement, h is the bilayer thickness, and αL is the average head group surface area per lipid. The lipid bilayer thickness was assumed to be 5 nm and average αL value for phosphatidylcholine, phosphoethanolamine and cholesterol were 0.65 ± 0.01 nm2, 0.52 ± 0.01 nm2, and 0.41 nm2, respectively.1 Using those values and the molar fraction of each component, the αL value obtained for our produced liposomes was 0.6 nm2/lipid. The Ntot was calculated to be 94771 lipid molecules per liposome of 100 nm. The number of liposomes per milliliter (Nlipo) can be derived from the lipid concentration with Avogadro’s number as shown in equation (2) =
× (2) × 1000
Where NA is Avogadro’s number, and Mlipid is the molar concentration of lipid. The number of liposomes was calculated based on the number of lipid molecules in a liposome and the lipid concentration used to fabricate the liposome solution. Briefly, the liposomes were analyzed with a dynamic light scattering particle analyzer (NanoFlex, Microtrac, USA). A mean diameter size of 81.9 ± 20.5 nm was determined. The calculation showed that the average encapsulated volume of a liposome is 3.8 x 106 nm3 (with an assumption that a lipid bilayer thickness is 5 nm for phosphatidylcholine and phosphoethanolamine). Given that the total concentration of lipid used to fabricate the liposomes is 84.6 µM, the number of liposomes Nlipo was calculated to be 5.38 x 1012 liposomes per mL. During the fabrication process, the liposome solution lost during extrusion and dialysis step was estimated to be around 10 ± 3% and 42.8 ± 2%, respectively. The data were evaluated preparing similar liposomes with fluorescent phosphatidylcholine lipids and analyzed with UV-vis spectrometry. Thus, the number of liposome recovery after preparation is calculated to be 2.77 x 1012 liposomes per mL.
2
Calculation of AuNPs aggregation degree in LAPIA assay Given that the plasmonic peak of aggregated AuNPs shifts to higher wavelengths (from 600 – 700 nm) depending on the aggregation degree, the most accurate way to analyze the data is to use UV-vis absorption spectrometry since it covers all the wavelength range. The microplate reader can only be used to measure the optical density at a single wavelength (655 nm). Hence, the results obtained with the microplate reader are only accurate when the maximum peak of aggregated AuNPs is at around 655 nm, which is the usual wavelength at which the optical density is obtained. At this wavelength, the results of the optical density obtained on the microplate reader are comparable to the absorption data obtained with UV-vis spectrometry. The AuNPs aggregation percent was calculated based on the peak area ratio of single AuNPs (520 nm) and aggregated AuNPs (from 600 – 700 nm) using the peak analyzer function in Origin software (Origin Pro v9.1, Originlab Corp., USA). The peak surface area ratio is used here to quantify the assembly degree of AuNPs. The ratio is normalized to express the assembly rate as a percentage change with the total assembly corresponding to 100%.2
Table S1. Characteristics of the produced liposomes and AuNPs, as measured by dynamic light scattering and TEM.
Sample
Particle size (nm)
Zeta potential (mV)
pH
AuNPs
15 ± 2
-68.1
5.9
Liposome-cysteine-PEG-biotin (100 nm)
82 ± 20
-36
4.0
Liposome-cysteine-PEG-biotin (200 nm)
242 ± 10
-30
3.9
Liposome-cysteine-PEG-biotin (400 nm)
355 ± 18
-26
4.1
3
Figure S1. Effect of pH on the ionization degree (%) of L-cysteine in water, based on its acidic (pK1 = 1.92) and basic (pK2 = 8.37) pKa values.
4
Figure S2. TEM image of PBS buffer with Tween 20 (0.05%) used to hydrolyze Cys-liposomes in LAPIA assay. The image does not show any vesicular structures, confirming the identity of the hydrolyzed liposomes in the TEM images of Figure 2c.
5
Figure S3. (a) Optical photograph of LAPIA test showing the effect of different concentrations of Cys-liposome on the aggregation of AuNPs. Liposome hydrolysis was caused by adding 20 µL PBST solution. (b) Correlation plot showing the aggregation degree as a function of Cysliposome concentration. The number of Cys-liposome was calculated based on their hydrodynamic diameter size and lipid concentration as mentioned above. AuNPs solution, adjusted to pH 7.25 using NaOH 0.5 M and 100 µL was added to each well followed by 40 µL of biotinylated Cys-liposome.
6
Figure S4. Schematic of the Liposome-Enhanced Nanoparticle Aggregation Immunoassay (LAPIA) for rabbit IgG detection. Difference concentrations of rabbit IgG were used as antigen. The reaction proceeds in different steps: (a) Sandwich capture of IgG target by capture anti-IgG immobilized on the plate and biotinylated anti-rabbit IgG antibody. (b) After incubation and washing, streptavidin is added to interact to biotinylated anti-IgG. (c) Biotin-conjugated Liposomes containing cysteine are added to the immunocomplex followed by AuNPs solution. (d) The addition of PBST-1X causes the breakdown of the liposomes and the release of cysteine, leading to immediate aggregation of gold nanoparticles and color shift from red to dark-blue (e).
7
Figure S5. Optical images of LAPIA plate showing the cross-reactivity in the detection of foodborne bacteria. Three microplates were coated with monoclonal anti-E.coli (A), antiSalmonella (B) and anti-Listeria (C). After the washing step, different concentrations of foodborne bacteria suspensions diluted in PBS-BSA 1% buffer solution, were added to the microplates. Specifically, Salmonella typhimurium, Listeria monocytogenes and E.coli O157:H7, were respectively incubated in microplates A, B and C. The detection was performed as previously described. The results clearly show high selectivity and absence of cross-reactivity on in the LAPIA test (the “blue spot” in (A) showed the false positive result in the LAPIA test).
8
Figure
S6.
Chemical
structure
of
L-cysteine,
L-α-phosphatidylcholine
(PC),
phosphoethanolamine-conjugated biotin used in liposome production, including 1,2-distearoylsn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000 labeled "PEG2000biotin").
References (1) (2)
Leekumjorn, S.; Sum, A. K. Biophys. J. 2006, 90, 3951-3965. Abbas, A.; Kattumenu, R.; Tian, L.; Singamaneni, S. Langmuir 2013, 29, 56-64.
9
