PerkinElmer Asperon Spray Chamber -Delivering Intact Individual

TECHNICAL NOTE

ICP - Mass Spectrometry

Patented Asperon Single

Cell Spray Chamber

Delivering Intact Individual

Cells to the ICP-MS Plasma

Introduction

Single Cell ICP-MS (SC-ICP-MS) is a technique that allows for

the analysis and quantification of metal content in an individual

cell. This technique can be used to quantify the uptake and

bioaccumulation of metals and metal nanoparticles in to cells,

as well as the intrinsic metals within the cells themselves, which

can be a measure of metabolomic changes related to cell stress

or lifecycle. SC-ICP-MS provides insights on:

• Metal content per cell

• Metal distribution in the cell population

• Number of cells containing metal or metal nanoparticles

• Number of nanoparticles per cell

PerkinElmer’s award-winning NexION

ICP-MS Single Cell Analysis

System offers a first-to-market complete solution to quantitatively

measure the metal content in individual cells, unveiling new

capabilities to study intrinsic metals content and the uptake of

dissolved (ionic) and nanoparticulate metals into cells, providing

new insights into drug delivery, mobilization/immobilization of

metal content, bioavailability, and bioaccumulation mechanisms.

The solution includes:

• Single Cell Micro DX Autosampler

• Single Cell Sample Introduction Kit with patented Aperon

™

spray chamber (US Patent No. 10,147,592)

• Syngistix

™

Single Cell Application Software Module

This technical note provides an overview of SC-ICP-MS system

validation methods:

• Optimization of sample flow rate for optimal cell transport

efficiency into the plasma

• Quantification of the mass of metal per cell using a standard

reference material

• Comparison of total digested metal in a sample compared to

manufactures data

For information related to sample preparation or workflow,

please consult our white paper "Single Cell ICP-MS Analysis:

Quantification of Metal Content at the Cellular Level".

Methods

ICP-MS Instrumental Conditions

All analyses were carried out on a PerkinElmer NexION

ICP-MS

series using two types of spray chambers: baffled cyclonic and

Asperon. The conditions used for both spray chambers are shown

in Table 1.

Parameter ICP-MS SC-ICP-MS

Spray Chamber Baffled Cyclonic Asperon

Nebulizer Glass Concentric

High Efficiency

Glass Concentric

Injector 2 mm Quartz

RF Power (W) 1600 W 1600 W

Spray Chamber Path Cyclonic Linear Pass

Neb Gas Flow (mL min

-1

) 0.92 0.32

Make-Up Gas (mL min

-1

) 0 0.7

Sample Flow Rate (mL min

-1

) 0.283 0.015

Sample Size Required

for Analysis (μL)

800-1000 100-200

Elements

140

Ce,

151

Eu,

153

Eu,

165

Ho,

175

Lu,

197

Table 1. ICP-MS Conditions for Acid-Digested Beads and SC-ICP-MS.

Polymer beads (Fluidigm, USA): Polymer beads laced with

lanthanide metals (

140

Ce,

151

Eu,

153

Eu,

165

Ho and

175

Lu) were

used as a suitable reference material due to the similarity in size

(2.5 µm) and density (1.05 g/cm

) to cells. The presence of

lanthanides in the beads enables them to be measured using

SC-ICP-MS. Although the size cut-off for the baffled cyclonic spray

chamber is in the 1-5 µm range,

2,3

the transport of these larger

beads (2.5 µm) is low, as illustrated later in Figure 2. The beads

were supplied at a concentration of 330,000 beads per mL and

diluted 10 fold for analysis. The mass of metal per bead can be

found in Table 2.

Gas and Sample Flows Optimization

The make-up and nebulizer gas flows and sample flow rate

had to be optimized to allow for equal transport of nano- and

micron-sized objects through the introduction system. Figure 1

shows the transport efficiency for nano- and micron-sized

objects through the introduction system for different sample

flow rates. It can be seen that as the sample flow rate decreases,

the transport efficiency for both nano- and micron-sized objects

increases and the difference between their transport gets less

pronounced with a transport efficiency of about 31% for both

the 60 nm Au NIST standards and the lanthanide-doped micron

beads (Fluidigm). The optimized conditions for this system can

be seen in Table 1.

Transport Efficiency Validation: Baffled Cyclonic vs.

Asperon Spray Chamber

A comparison study of the transport efficiency of nano- and

micron-sized particles between the baffled cyclonic and Asperon

spray chambers is shown in Figure 2. The systems were both

optimized for maximum intensities while keeping oxide and

double-charge formation below 2.5% (Table 1). It can be seen

in Figure 2 that both introduction systems transport a sufficient

amount of NPs (NIST 8013 60 nm Au NPs) into the ICP-MS to

provide a statistically significant measure of the mean and

standard deviation of the NPs (either in diameter or mass per NP)

and particle number concentration showing that both systems

work equally well for nanoparticles. However, the number of

micron-sized particles transported by the baffled cyclonic spray

chamber is low, with only a few beads being analyzed compared

to the Asperon spray chamber, where a significant number of

beads were counted.

Standards for SP-ICP-MS and SC-ICP-MS: Standards of 1, 2

and 3 ppb

140

Ce,

151

Eu,

153

Eu,

165

Ho and

175

Lu were prepared

for measuring the mass of metals in the polystyrene beads.

All standards for SC-ICP-MS were prepared in ultra-pure water.

The transport efficiency was determined with the 60 nm Au

NPs (NIST 8013) at a concentration of 50,000 part. mL

-1

Digestion of polystyrene beads: Digestion was accomplished

by placing 5 mL of the bead suspension into a PTFE digestion

vessel with 5 mL of hydrogen peroxide (Optima grade) and

10 mL of nitric acid (Optima grade). The mixture was allowed to

sit for 10 minutes to allow gasses to be released from any initial

reactions before the vessels were sealed. The beads were then

digested in a Titan MPS

™

Microwave (PerkinElmer), following

the program in Table 3. After they had cooled, the samples were

diluted to 2% acid for analysis. Standards of 10, 50, 100 and

200 ppb Lu, Eu, Ho and Ce were matrix-matched to the samples

for ICP-MS analysis. Ge and In were spiked into the samples as

internal standards.

Element Atoms Per Bead (± 15%) Mass (ag) (± 15%)

Ce 140 19.9E6 4626.26

Eu 151 11.3E6 2833.38

Eu 153 12.0E6 3048.75

Ho 165 7.6E6 2082.32

Lu 175 9.8E6 2847.82

Table 2. Supplier information on lanthanide laced polystyrene beads.

Stage

Temperature

(°C)

Pressure

(bar)

Ramp

(min)

Hold

(min)

(%)

1 150 70 2 5 60

2 190 75 2 10 80

3 210 80 2 15 90

4 50 80 1 10 0

5 50 0 0 0 0

Table 3. Conditions for microwave acid digestion of polystyrene beads.

Figure 1. Optimization of sample ow rates through the introduction system to maximize

the transport of nano- and micron-sized objects into the plasma.

0 500 1000 1500 2000 2500 3000

Transport efficiency %

Particle size (nm)

TE for nano- and micron-sized particles at

different sample flow rates

15.23

17.08

20.95

38.5

52.35

Sample flow

rate µm/mL

Figure 2. Real-time screen shots comparing the number of Au NIST 60 nm NPs and 2.5 µm polystyrene beads making it to the plasma for the baed cyclonic and Asperon spray chambers.

Asperon™

60 nm Au NIST

2.5 μm Beads

Baffled Cyclonic

60 nm Au NIST

2.5 μm Beads

As shown in Table 4, the transport efficiencies for the 2.5 µm

polystyrene beads and 60 nm Au NPs in the baffled cyclonic spray

chamber are 0.04 ± 0.02 % and 2.42 ± 0.06 %, respectively, which

is significantly different (calculated probability (p) < 0.05, Student's

t-distribution). This translates to approximately 3.5 ± 1.9 beads per

minute reaching the plasma in a suspension containing 33,000

beads mL

-1

and 342.3 ± 8.7 NPs per minute for a suspension

containing 50,000 NPs mL

-1

. Clearly, the cyclonic spray chamber is

not suitable for single cell analysis. In contrast, the Asperon spray

chamber has a transport efficiency which is not significantly different

for the beads or the NPs: 31.33 ± 2.54 % and 30.31 ± 1.85 %,

respectively. This corresponds to 229.5 ± 19.5 NPs and 208.3 (± 12)

micron-sized beads for suspensions containing 50,000 particles mL

-1

and 33,000 beads mL

-1

respectively.

Quantitative Validation: Measurement of Metal Mass

per Bead

The polymer beads are doped with lanthanide metals (

140

Ce,

151

Eu,

153

Eu,

165

Ho and

175

Lu) at the concentrations shown in Table 5, as

Spray Chamber

Baffled

Cyclonic

Asperon

TE 60 nm NIST (%) 2.42 (±1.85) 31.33 (± 2.54)

Number of 60 nm NIST NPs

Measured in a One-Minute Scan

342.3 (± 8.7) 229.5 (±19.1)

TE 2.5 μm beads (%) 0.04 (± 0.02) 30.31 (±1.85)

Number of 2.5 μm Beads

Measured in a One-Minute Scan

3.5 (± 1.9) 208.3 (±12)

Table 4. A comparison of the number of nano- and micron-sized particles entering

the ICP-MS and subsequent transport eciencies.

supplied by the manufacturer. The mean mass of metal per bead

as measured by SC-ICP-MS can also be seen in Table 5. Comparing

the experimental data with the supplied concentrations, there is

no statistical difference (all p values >> 0.05) between the mean

mass provided by the supplier and those quantified using SC-ICP-

MS. The number of beads per mL was measured to be 34,821

(± 661.7) having no statistical difference to the manufacturer's

value of 33,000 (± 1650).

Element Data Provided by Supplier Measured by SC-ICP-MS (Ag Per Bead)

Atoms Per

Bead (±15%)

Mass

(ag)

Size

(nm)

1 2 3 4 Average STDEV

Ce 140 19.9E6 4626 109.7 4250 4561 4750 4699 4632 95

Eu 151 11.3E6 2833 101.1 2961 3012 3123 3075 3043 61.6

Eu 153 12.0E6 3049 103.6 2904 33240 3114 3061 3080 120.4

Ho 165 7.6E6 2082 76.8 2157 2228 2576 2354 2329 159.3

Lu 175 9.8E6 2848 82.1 2921 2228 2965 2982 2889 68.8

Number of

Particles

34323 33145 34696 34821 34246 661.7

Table 5. Metal Content per Bead and Bead Concentration: A Comparison of SC-ICP-MS vs. Certicate Values.

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The total metal content of the bead suspension was established

after acid digestion and was found not to be statistically different

from those calculated from the mass per bead and bead number

measured by SC-ICP-MS (Table 6).

Element

Values From

Supplier (ppb)

Measured

Values (ppb)

Ce 140 152.67 160.3 (7.4)

Eu 151 93.50 71.6 (7.6)

Eu 153 100.61 80.6 (8.0)

Ho 165 68.72 67.8 (8.4)

Lu 175 93.98 83.9 (8.1)

Table 6. Total amount of metal in bead suspension aer acid digestion.

References

1. Single Cell ICP-MS Analysis: Quantification of Metal Content

at the Cellular Level, PerkinElmer Inc., 2017.

2. Schaldach, G.; Berger, L.; Razilov, I.; Berndt, H.,

Characterization of a cyclone spray chamber for ICP

spectrometry by computer simulation. Journal of Analytical

Atomic Spectrometry 2002, 17, (4), 334-344.

3. Matusiewicz, H.; Slachcinski, M.; Almagro, B.; Canals, A.,

Evaluation of Various Types of Micronebulizers and Spray

Chamber Configurations for Microsamples Analysis by

Microwave Induced Plasma Optical Emission Spectrometry.

Chemia Analityczna 2009, 54, (6), 1219-1244.

SC-ICP-MS Components

Component

Part Number

NexION 1000/2000

Part Number

NexION 300/350

Single Cell Micro

DX Autosampler

N8150039 N8140039

Single Cell Sample

Introduction Kit

with Asperon

Spray Chamber

N8150032 N8140032

Syngistix Single

Cell Application

Software Module

N8150321 N8150321

Conclusion

This work has shown that the patented Asperon spray chamber

provides increased transport of micron-sized objects into the

ICP-MS compared to traditional introduction systems. Coupled

with the fast data acquisition capabilities of the NexION ICP-MS,

the Asperon

spray chamber has allowed for the quantification of

masses from micron-sized objects down to the attogram per-bead

level as well as providing accurate measurements of the number

concentration of the beads per milliliter.