# Presentation: 407484

## MXRF-Based Molecular Recognition Technology

- Benjamin Warner, George Havrilla, Thomasin Miller,Cyndi Wells
- Chemistry Division, and
- Non-Proliferation and International Safeguards Division
- Los Alamos National Laboratory

## X-ray fluorescence:

_**X-ray fluorescence:**_
- Detect and quantify elements by measuring wavelengths and intensities of characteristic emissions
_**Advantages:**_
-  Non-destructive
-  No sample preparation
-  Elemental analysis
-  “Tags” not necessary
-  Nanogram detection
-  Screens 15 compounds/sec

## Technology

- Previous reports stated that a synchrotron was necessary for
- Sufficient resolution
- Sufficient beam intensity, especially to penetrate capillary
- Our results include
- Bench-top microfocus instruments have sufficient resolution, which is less than the nominal spot size
- Bench-top microfocus instruments have sufficient intensity to penetrate capillaries
- Detection limits are on the sub-nanogram level

## Technology

- We attempted the following reaction (Berkessel and Herrault, ACIEE, 1999, 38, 102 – 105):

- LANL

- Berkessel

- EPPS, H2O

## Bead 13: H2N-Ser-Gly-Gly-His-Gly-Gly-Arg-Gly-Gly-His-Phe-CO2H

- Bead 13: H2N-Ser-Gly-Gly-His-Gly-Gly-Arg-Gly-Gly-*His*-Phe-CO2H
- Bead 10: H2N-Ser-Gly-Gly-His-Gly-Gly-Arg-Gly-Gly-*Arg*-Phe-CO2H

## Zr

- 1

- 4

- 7

- 10

- 13

- 2

- 3

- 1

- 4

- 7

- 10

- 13

- 1

- 4

- 7

- 10

- 13

- Br

- P

## Visual Method:

- 1)  Little information on binding :
- peptide only
- peptide + substrate
- peptide + substrate + metal
- All three possibilities result in clear beads.
- 2)  Subjective interpretation:
- i.e. dark blue vs. light blue
- 3)  Activity of the catalyst:
- only know product

- MXRF Method:
- 1)  Full information on binding :
- peptide only
- peptide + substrate
- peptide + substrate + metal
- Can differentiate and quantify.
- 2)  Quantitative interpretation:
- i.e. quantify amounts of each element
- 3)  Activity of the catalyst:
- TON: product to catalyst ratio

## Br

- S, from EPPS

- P

- Ca

- Zr

- EPPS

## Slide 9

## Slide 10

## Slide 11

## Rh X-ray

**Rh X-ray**
**tube**

**Detector**

**XYZ stage**

**Interlock**

**Power Supply**

**MXRF sample chamber**

**Detection **
**Window**

**CE **
**Apparatus**

## Slide 13

## Styrofoam support

**Styrofoam support**

**Buffer **
**vial**

**Interlock**
**Switch**

**Capillary**

**Pt Electrode**

**Latch**

**From**
**Power supply**

## Selection of Capillary

*** Based on sample volume assuming a 50 ********m MXRF spot size**

| SiO2 Tubing  | Dimensions |  |  | Detection Limit (mMolar)* |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | O.D. (m) | I.D. (m) | Wall Thickness (m) | Cu | Zn | Fe | Co |
| TSP167100 | 164 | 97 | 33 | 1.32 | 2.35 | 1.58 | 1.90 |
| TSP250350 | 362 | 256 | 53 | 0.72 | 1.10 | 0.16 | 0.53 |
| TSP530660 | 666 | 534 | 66 | 0.13 | 0.32 | 0.18 | 0.42 |
| TSP075375 | 363 | 75 | 144 | 31.23 | 7.46 | 16.76 | 21.19 |

**Higher energy X-rays allow detection of species through thin-walled fused silica capillary**

## Slide 16

## Aqueous Separation of Co2+ and Cyanocobalamin (Vitamin B-12)

**Co****2+**

**Vitamin B-12**

**3.4 mMolar Co****2+**** and 10.2 mMolar Cyanocobalamin in de-ionized H****2****O**
**70 cm capillary (100 ********m id, 170 ********m od)**
**75 mM Trizma run buffer, pH 8.0**
**4 s pressure injection at 380 mbar**
**10 KV running potential**

## Separation of Ferritin and Cyanocobalamin (Vitamin B-12)

**Ferritin**

**Vitamin B-12**

**1.16 mg/mL Ferritin********* and 10.2 mMolar Cyanocobalamin in de-ionized H****2****O**
**70 cm capillary (100 ********m id, 170 ********m od)**
**100 mM Trizma run buffer, pH 8.5**
**4 s pressure injection at 253 mbar**
**9.5 KV running potential**

## Common CE Elemental Detection Methods

## Deposition Reproducibility with Nanoliter Injector

**50 nL**

**10 nL**

**5 nL**

**2 nL**

**Deposition onto AP1 film produced the most reproducible spots.**

- All spots were deposited with a Nanoliter Injector A203 (WPI Inc.)

## Effect of Multiple Elements on the Intensity of a Single Element in a Dried Spot

**5 ng Cu**

**5 ng Cu + 2 elements**

**5 ng Cu + 4 elements**

**5 ng Cu + 9 elements**

**All spots were dried on AP 1 Film using the UltraMicro Pump II Microliter injector (WPI Inc.).**

## Effect of Multiple Elements on the Intensity of a Single Element in a Dried Spot

## Constant Volume Spots, Variable Mass Deposited

**Cu intensity increases with mass deposited**
**Precision decreases with increasing mass deposited**

## Detection Limit of Cu for Given Dried Spot Volumes

|  | Cu Detection Limit (ng) |  |
| --- | --- | --- |
| Volume of Spot Deposited (nL) | AP1 | Polypropylene  |
| 2 | 0.1 | 0.4 |
| 5 | 0.2 | 0.6 |
| 10 | 0.4 | 0.8 |
| 50 | 1.8 | 2.6 |

- All spots were deposited using a Nanoliter Injector A203 (WPI Inc.).

**Lower detection limits achieved with AP1 film**
**Detection limits decrease with smaller spot volume**

## Constant Mass Spots, Variable Volume Deposited

**Cu intensity decreases with increasing spot volume for a given mass**
**Smaller spot volumes allow for better mass detection**

## Effect of Spot Volume on Drying Pattern

**1000 nL**

**10 nL**

_**Cu Concentration (mg/L) Deposited **_

**10 mg/L**

**100 mg/L**

**1K mg/L**

**10 K mg/L**

_**Spot**_
_**Volume**_

**Drying pattern of smaller droplets is more circular and uniform**
**Less concentrated spots are most affected by larger spot volumes**

**Effect of Spot Volume on Drying Pattern**

## Effect of X-ray Beam Size on Apparent Dried Spot Size

**Spots were deposited with a ****10 ********L** **transfer pipette on polypropylene film**

**70 ********m **

**X-ray Beam Diameter:**

**200 ********m **

**350 ********m **

**Effect of X-ray Beam Size on Apparent Dried Spot Size**