Development and optimization of heavy metal lead biosensors in biomedical and environmental applications : Journal of the Chinese Medical Association

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Development and optimization of heavy metal lead biosensors in biomedical and environmental applications

Chang, Tai-Jaya,b; Lai, Wei-Qunc,d; Chang, Yu-Fene; Wang, Chia-Lina,b; Yang, De-Mingc,d,*

Author Information
Journal of the Chinese Medical Association: August 2021 - Volume 84 - Issue 8 - p 745-753
doi: 10.1097/JCMA.0000000000000574
  • Open

Abstract

1. HIDING DANGER OF LEAD

Lead (Pb), as a heavy metal, has been used by humans in various fields. For example, it can be used as the protective apron for radiation shielding, and can be use as the adulteration ingredient for sweet-tasted wine in the history of ancient Rome Empire.1 For more than 70 years, Pb-containing water pipelines have been used in Taiwan for various purposes. Now, Pb-contaminated drinking water exist in many countries and Pb-contaminated irrigation water in some countries.2,3 However, Pb-containing materials have been used by humans for some special purposes, such as in the traditional Chinese medicine, in the ingredients for painting the wall and glasses, in hair dyes, and in toys’ coloring. The lead content in Petrol and Diesel are used in some urban environments to balance the outcome between the amount of Pb-absorbed from environmental exposure and the health deficits occurred due to less notice of Pb, as reported by Dr. Clair C. Patterson.4 Dr. Patterson was the pioneer to estimate the aging of the Earth by determining the isotopic composition of Pb.5 His findings reported on the detrimental impact of Pb released from Pb-containing gasoline into the air we breathe. However, researchers took a long time to become aware of the horrific impact of Pb on humans (Table 1). Without significant symptoms found under chronic poison of low-level Pb13, the leaded gasoline would be used freely without control, and safety regulations are required in our environment.14

Table 1 - Selected events of Pb poisoning with significant symptoms
Years Area Source Symptoms Ref
1994 Michoacán, Mexico Ceramic folk-art Renal, reproductive, neuromuscular dysfunctions, behavior alterations in children, etc. 6
2004 Guangdong, China Electronic waste Skin damage, headaches, vertigo, nausea, chronic gastritis, gastric ulcers, etc. 7
2008 Shaanxi, China Metal smelting Abdominal pain, developmental delay, irritability, etc. 8
2010 Zamfara, Nigeria Mining Seizures, hearing problems, irritability, etc. 9
2012 Hunan, China Chemical plant Developmental delay, memory loss and abdominal pain in children, etc. 10
2013 Kabwe, Zambia Lead–zinc mine Central nervous system damages, etc. 11
2020 Taichung, Taiwan Traditional Chinese medicine Abdominal pain, insomnia, etc. 12

2. KNOWING EXPOSURE STATUS OF Pb

Nowadays, regulations for preventing the invasive toxicity of Pb to humans have been set up by World Health Organization (WHO) and by many developed countries (Table 2). Briefly, the test of blood lead level (BLL) is so far the only effective way to understand the status of Pb exposure in human body (Fig. 1). BLL represents the amount of Pb detected in the blood. According to previous evidence found from Pb-affected patients (adults or young children) with various symptoms, BLL is officially suggested not to exceed 10 μg/dL in adults and should be <5 μg/dL in children (Table 2).15,16 More recently, Pb content in urine or serum was also used in toxicity diagnosis alternatively. However, the standard values for safe permissible levels of urinary/serum Pb levels are yet to be determined. Followingly, the observation of possible entry routes for Pb such as drinking water and intake of foods was made. The permissible concentration of Pb in tap water, foods, and mushroom (dry weight) are set at 7 ppb (0.7 μg/dL, Taiwan CNS 8088) or 10 ppb (1 μg/dL, WHO 2017), 300 μg/Kg (30 μg/dL), and 3000 μg/Kg (300 μg/dL), respectively (Table 2).17

Table 2 - Regulations which limit the contents of Pb within blood of human or detected in the water, or foods
Standard/unit conversion ppb (μg/L) μg/dL nM
Blood lead level (BLL) for adult 100 10 500
BLL for children 50/25 5/2 250/100
WHO 2017 Pb in tap water 10 1 50
CNS 8088: Pb from faucet Taiwan 7 0.7 35
Food containing Pb 300 30 1500
Mushroom containing Pb 3000 300 15 000
BLL = blood lead level.

F1
Fig. 1:
Measurement procedure of Pb content extracted from environment or human body.

Many issues need to be overcome in the examination of Pb concentration from blood (BLL) or from other tested targets (water or foods—the ingestion sources). For example, Pb reagent preparation requires the use of strong acid and base, which need to be handled with care to avoid the risk of occupational disaster. In addition, it requires professional training for personnel to operate the precision instruments (eg, atomic absorption spectroscopy or inductively coupled plasma mass spectrometry). Of course, gaining Pb-content data using the whole complicated procedure is time-consuming. Finally, such tests can be carried out only in limited places, either in hospitals (blood drawing) or special companies equipped with atomic absorption spectroscopy or inductively coupled plasma mass spectrometry, and needs specialists for operating the equipment (Fig. 1).

Through long-term observation, scientists gradually proposed that no safe BLL exists, if safety is defined as the level not harmful to human life.18 In fact, chronic exposure to even lowest BLL (as low as 2 μg/dL) in children has been confirmed to possibly lead to various kinds of neurodevelopmental impairments, ranging from permanent cognitive damages to numerous neurodegenerative diseases, without specific behavioral alterations or clear significant symptoms.19 Furthermore, low-level Pb exposure was also confirmed to be a risk factor that contributes to cardiovascular disease and increases the overall mortality rate, once entering and staying in human body (Table 3).27 Recent studies from Taiwan also reported on the association of urinary Pb with cardiovascular disorder (by measuring the thickness of carotid intima-media) and with metabolic syndrome in young generations.28,29 Thus, the toxicological mechanisms at very low contents of Pb exposure need to be urgently explored, especially in young populations. In addition to BLL, knowing Pb contents within the living body is another challenge for understanding more about the toxicology of the heavy metal Pb.

Table 3 - Selected historical events for low BLL induced defects (<5 or even 2 μg/dL)
Years Affect Ref
2000 A small increasing in the number of red blood cells and in girls with reducing mean corpuscular volume and mean corpuscular hemoglobin. 20
2006 Irregular menstruation, Increasing the risk for infertility. 21
2007 Correlated to simple reaction time that reflects attention (p = 0.05). and digit span (p = 0.08). 22
2012 A higher semen lead concentration was correlated with lower sperm count. 23
2014 Decreasing birthweight and increasing the odds of preterm birth among boys. 24
2017 An increasing risk of dental caries of the deciduous teeth 25
2017 Correlated positively with red cell distribution width; and negatively with child size, age, body mass index, hemoglobin, platelet distribution width, gamma-glutamyl transferase (γ-GT) and IQ. 26
BLL = blood lead level.

3. BIOSENSORS TO EXPLORE THE SECRET OF LIFE

The fluorescent biosensors (FBs) in various forms (i.e. either chemical indicators or genetically-encoded [GE] fluorescent protein [FP] biosensors [GEFBs, Fig. 2]) that are compatible with a spectral/signal recorder or a fluorescent microscope can be used for the real-time detection of specific targets whether extracted from environments or tested inside living body.31 By applying such FBs, the content dynamics of a targeted molecule within or even outside the living body can be directly detected and shown at the aspects of time and space. The functions of the probed interests can be further understood through the help of these GEFBs.

F2
Fig. 2:
Design of genetically-encoded fluorescent biosensors (GEFBs). A, Single fluorescent protein biosensor will proceed conformational changes after target-sensor binding. B, The fluorescent emission spectra of such a single-FP biosensor will either in increase (on, ECFP Blue) or decrease (off, EYFP Yellow) mode. C, Fluorescent energy resonance transfer (FRET)-based biosensor uses two FRET FP pair proteins, either EBFP with EGFP or ECFP with EYFP. D, Conformational changes happen when target-sensor binding exists. The fluorescence intensity (FI) of EGFP (or EYFP) increases, and then EBFP (or ECFP) decreases. C, Graph was adapted from previous report.30

The concept for probing interested targets by GEFBs is adapting the sensing key as a specific receptor within the FP domain, either inside single FP biosensors for the conformational changes (Fig. 2A, B)31 or between the two FP pairs for the reaction of fluorescence resonance energy transfer (FRET, Fig. 2C, D).30 In single FP biosensors, the fluorescent intensity (FI) of single FP increases to turn “on”, or decreases to turn “off” after receptor-target recognition-binding, when sensor-target exists (Fig. 2B). In FRET-based biosensors, such receptor-target binding within FRET pairs generate FRET signals (Fig. 2D). In both ways, the sensing work can be accomplished.

“Cameleon”30 is the first GEFP biosensor borne in 1997 by Prof. Roger Tsien, who won the 2008 Nobel Prize for Chemistry. This biosensor monitors intracellular calcium (Ca) ions through acquiring event signals of FRET between 2 FRET pair FPs.30 Such brilliant concept has been proved to be workable and allowed measuring the dynamics of intracellular targeted signals inside living cells in a time-lapse manner, and alternatively allowed amplifying chemical indicators, which needs an additional preloading procedure. Following Cameleon, more than 50 kinds of FRET-based or similar biosensors were developed continuously (Table 4). These tools can help scientists to observe certain conditions of living cells such as the oncogenetic processes of tumors68 and even to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).67 To build up metal ion FBs or GEFBs (eg, lead [Pb],69 cadmium [Cd],70 silver [Ag],71 copper [Cu],72 or zinc [Zn],63,73 etc], more criteria like molecular selectivity are required. Thus, developing GEFBs for detecting metal ions is relatively hard and therefore is less to be seen.

Table 4 - Examples of FRET-based biosensors
Application Examples Mechanism Sensory key(s) FRET pair Ref
Protein binding interaction Multimerization of IL-17RA Inter IL-17RA with itself CFP YFP 32
GPCR subunit association Inter Gα with Gβγ CFP YFP 33
Transcriptional factor Erg and Jun interaction Inter Erg with Jun CFP YFP 34
Protein conformational change Sensing membrane potential Intra S Potassium channel voltage sensing domain ECFP EYFP 35
GTPase Activation and signaling of rac and cdc42 Intra M or Intra S Cdc42 or rac with GTPase binding domains CFP YFP ECFP EYFP 36, 37
Protease activity Caspases Cleavage Caspase proteolytic substrate CFP YFP Cerulean Venus 38–42
Calpain Cleavage Calpain proteolytic substrate ECFP EYFP 43
Factor Xa Cleavage Factor Xa proteolytic substrate BFP5 RSGFP4 44
Kinase/phosphotase activity MLCK and MLCP Intra S RMLC (regulatory myosin light chain) ECFP Citrine 45
Kinetics and potencies of 12 known PKC ligands Intra S PKCδ ECFP EYFP 46
Detection of PKC activities Intra S Truncated pleckstrin containing PH and DEP domains ECFP EYFP 47
Phosphorylation by insulin receptor Intra M Phosphorylation recognition domain and its binding substrate CFP YFP 48
Activities of EGFR, Src and Ab1 Intra M SH2 with phosphorylation substrates for EGFR, Src and Ab1 CFP YFP 49
Activation of Src Intra M SH2 with phosphorylation substrates for Src CFP YFP 50
Metabolic molecules Glucose Intra S Glucose binding protein ECFP EYFP 51–53
Maltose Intra S Periplasmic binding proteins ECFP EYFP 54
Glutamine Intra S Glutamate/aspartate binding protein ybeJ ECFP Venus 55
Signalling molecules cAMP Inter PKA with cAMP-dependent binding substrate CFP YFP 56
IP3 Intra S InsP3 receptors CFP YFP 57, 58
cGMP Intra S GKI and PDE CFP YFP 59
Estrogen receptor ligand Intra S Estrogen receptor ligand binding domain CFP YFP 60
Ca2+ in ER Intra S apoK1-er CFP YFP 61
Ca2+ Intra M CaM M13 CFP YFP BFP GFP 30
Zn2+ Intra M Atox1 WD4 CFP YFP 62, 63
ATP Intra M ε subunit of the bacterial FoF1-ATP synthase. CFP Venus 64
Other molecules Specific RNA sequence Intra S HIV-1 Rev protein ECFP EYFP 65
SARS CoV-2 Spike protein Intra M hACE2 Cy3 Cy5 66
SARS CoV-2 Cleavage 3-chymotrypsin-like cysteine protease (3CLpro) substrate ECFP Venus 67

4. CHEMICAL Pb BIOSENSOR

Idealistically, we live in a relatively healthy place if there is no violation against the law, no unscrupulous adding toxic stuffs into the food, water or in the air. However, the real world is somehow we need to be precocious about resources with more abnormal ingredients around our environment on purpose. We should take more caution to survey convenient and precise methods to verify the contents of toxicants such as the heavy metal Pb we intake incidentally. Taking advantage of drinking or even breathing through any kind of perception technology is available from specialized hospitals or companies (Fig. 1). For precisely understanding the environmental (outside human body) and poisoned (inside human body, BLL, or in vivo) status of heavy metal Pb and monitoring their lethal contents will be direct and efficient on the aspect of source/absorbance control.74,75 The sensor methods and required regulations for the so-called accepted amounts of Pb in certain targets should be solid and confirmed, as mentioned in Table 2.15–17

To deal with the Pb issue, we had previously applied a kind of chemical indicator indo-1 (originally for Ca) as a novel Pb sensor.76 The crucial point of indo-1 being able to sense Pb is that Pb can specifically quench the fluorescent intensity (FI) of indo-1 at spectral measurement around 450-470 nm (Fig. 3A). FI of indo-1 is Ca-insensitive at 440-450 nm (Fig. 3B).77 With this chemical indicator, we provide evidence that Orai1 with STIM-1 as a kind of store-operated calcium channels (SOCs) plays a dominant role in cytosolic Pb entry (Fig. 3C-E).76,78 It seems to be relatively convenient using indo-1 as an alternative method to measure the existence of Pb, although this chemical probe has many drawbacks. The first drawback is that the photo-instability of indo-1 causes the photo-toxicity and even photo-activation of the reagents within the tested cells. Second, due to the weak FI of indo-1, the cell-loading procedure takes more time, with an extra problem in difficulty distinguishing the reduced FI signals from the illumination-induced photo-bleaching and the Pb-dependent photo-quenching. Third, none of the chemical probes can be trapped into specific subcellular compartment to sense target molecule at present. The fourth key point is the cost. Such kinds of chemical probes generally cost high, and they require relatively a large amount for dye loading and the following sensing processes.

F3
Fig. 3:
The use of chemical fluorescent sensor indo-1 to detect intracellular entry of Pb from extracellular environment. A, The fluorescent emission spectra of indo-1 at different concentrations of Pb (upper) or those of Ca (lower). Pb can quench the fluorescent signals of Indo-1 at around 450 to 470 nm (red arrows shown in upper part), and this wavelength area is almost Ca-insensitive (red dash line shown in the lower part). These figures are originally from Legare et al77 and permission has been obtained to use the same here. B, Functional role of store-operated Ca channel (SOC, composed by a membrane channel Orai1 shown with green fluorescence, and an ER membrane protein STIM1 shown with red fluorescence) for the intracellular entry of Pb probed by indo-1 (shown with blue fluorescence) using different types of cells (upper: PC12; lower: HeLa). Right: The time-lapse recordings of indo-1 at different conditions—for example, control (Ctl), activated SOC; SOC blocker 2-APB; activated SOC with SOC blocker. The data are originally from Chang et al,76 and permission has been obtained to use the same here. C, Further evidence on the role of SOC through overexpression of Orai1 and STIM1. Left: Confocal images of Orai1 (green) and STIM1 (red) is shown in the localization of them. Right: The time-lapse recordings of indo-1 at different conditions. The data are originally from Chiu et al78, and permission has been obtained to use the same here.

5. FRET-BASED Pb BIOSENSORS

Since we did not have much experience on constructing GEFBs previously, it was indeed a great challenging task to develop Pb GEFBs. Thanks to Prof. Roger Tsien for giving us personal encouragement and suggestions in early 2008 before he gained the Nobel Prize. In 2012, we made the first version of FRET-based Pb biosensor Met-lead 1.59, so that the in-cell content monitoring of Pb can finally be done alternatively.69 PbrR (a novel Pb binding protein) was selected as the Pb-sensing key within Met-leads. PbrR79 was originally found from a special bacteria Cupriavidus metallidurans (CH34),80 which helps the organism to survive longer in the waste water of factories. The major functional domain of PbrR was cloned and re-ligated into the backbone of YC3.6 (replacing the Ca sensing motif: calmodulin and M13)81 to form Met-leads (molecular structure proposed in Fig. 4A). Finally, the performance of fluorescent spectral Met-lead (Fig. 4B) provides a direct evidence to demonstrate the FRET signal manipulation (functional Pb sensing) when Pb exists.

F4
Fig. 4:
Design, spectra, and performance of FRET-based Pb biosensor Met-lead. A, FRET design of Met-lead. B, The spectra of Met-lead shown FRET event could happen when Pb exists. C and D, Time-lapse record of Met-lead displays the emission ratio increase when Pb exists with (C) or without ionomycin (D). The data shown in (A and B) and (C and D) are originally from Yang et al82 and Chiu and Yang69, and permission has been obtained to use the same here.

Discussing about sensor ability of the first version of Met-lead 1.59 (ie, the dynamic range [DR] and the sensitivity [limit of detection, LOD]), the DR is less than 2-fold (emission ratio from 3.3 to 5.7; Fig. 4C), and the practical LOD of Met-lead 1.59 is 100 nM (~2 μg/dL) or 500 nM (~10 μg/dL) with or without ionophore (ionomycin), respectively (Fig. 4D).69 The sensing ability of Met-lead 1.59 was obviously not fully qualified for further real applications (compared with the regulation required in Table 2), although it was a very good start for the development of FRET-based Pb biosensor. For the specificity of Met-lead 1.59, the ionic selectivity of Met-lead 1.59 has been tested on various ions (eg, Ca, Mg, Mn, Fe, Cu, and Zn). The only interfered ions are Cu and Zn.69 In addition to the FRET-based Pb GEFBs, we also developed a FRET-based cadmium (Cd) biosensor by applying CadR as the sensing key.70

6. OPTIMIZATION OF FRET-BASED Pb BIOSENSORS

As described above, the low level of Pb is indeed quietly threatening human health without apparent signs of considerable dangers. Due to the relatively low DR (less than 2-fold) and sensitivity/LOD (only fare for adult BLL level: ~10 μg/dL) is not well-verified for children’s limit 5 μg/dL. Met-lead 1.59, as the first version of FRET-based biosensor, was not a good for further Pb biosensing.69,82 Therefore, we tried to improve the sensing ability of Met-leads through different ways. First, utilizing the original structure of PbrR with six α-domains83 to let us consider the adjustment of PbrR in lengths (different number of α-domains) may change the space distance between the two FRET pair FPs to modify the sensing level of Met-leads upgraded.69,84 Second, dimerization of PbrR via constructing a three-cysteine Pb-binding socket is required to sende MerR-like protein family.83 As the multiple-meristic property could cause functional instability of Met-leads, it could be possible to break in a such multimer by inserting a repeat sequence (linker) within the middle position of PbrR. Actually, the sensing ability of Met-leads will be improved alternatively afterward.84

So far, Met-lead 1.44 M1 is the optimized version with the best DR (almost 5-electronic fold, Fig. 5A) and LOD (10 nM, 2 ppb; Fig. 5B).84 The dramatically expanded DR of Met-leads led us to explore the basic Pb toxicological researches involving in vivo biosensing (eg, on live species such as Drosophila and Arabidopsis (Fig. 5A), respectively).78 Newly-developed Met-lead 1.44 M1 with a high sensitivity (Fig. 5B) is five times lower than WHO-permitted level for tap water (10 ppb, Table 2) and 50 times lower than the BLL for children, 5 μg/dL (50 ppb, Table 2). Thus, Met-lead 1.44 M1 has met many important potential practical needs: the Pb detections from environment (in vitro, drinking or irrigation water) or body fluid (in-cells, serum or urine), and others (in vivo, whole animal or plant), which has been widely well-noticed in researches.

F5
Fig. 5:
Sensing ability of optimized FRET-based Pb biosensor Met-lead 1.44 M1. A, The dynamic range (DR) of FRET-ratio changes is up to 460% (within cotyledons of Arabidopsis seedlings). B, The limit of detection (LOD) is 10 nM (2.0 ppb). The data are originally from Yang et al,82 and permission has been obtained to use the same here.

7. FUTURE PERSPECTIVES

Scientists have tried to combine 3C electronics such as smart phone to construct easy-to-use biosensors.84–94 Such portable devices would gradually become popular because of having a new advanced camera. Through visible light information or fluorescent signals, the mobile-tools can achieve good sensing abilities either using cuvettes or plates/microfluid camber to support target sources. Thus, it would be a great task to combine smart-phone with Met-leads to construct a new portable FRET-based sensing device in the future (Fig. 6). The new easy-to-handle device containing a biosensor-chip like Met-leads will allow us to imply real-time, and to precisely measure the contents of Pb everywhere, such as in tap or irrigation water, human bloods/serums or urines, etc. (Figs. 1 and 6). Meanwhile, the single FP-based Pb biosensors (Fig. 2A, B) can be more conveniently applied than FRET-based biosensors (because FRET-based biosensors occupy two fluorescent channels,95 but single FP-based biosensors needs only one), and can be formulated as per the guidance of molecular simulation in the future (for examples of animated Met-lead, visit https://reurl.cc/ygb4ny).78

F6
Fig. 6:
The integration of various Pb detection methods into a portable device.

ACKNOWLEDGMENTS

This work was supported by Ministry of Science and Technology of Taiwan (MOST 108-2745-8-075-001-, MOST 105-2320-B-075-002, NSC-97-2320-B-075-005-MY3, NSC-100-2320-B-075-004, NSC-102-2320-B-075-002) and Taipei Veterans General Hospitals (V98C1-052, V99C1-002, V100C1-032, V101C1-072, V102C-163, VGHUST102-G7-1-2).

We thank Professors Daniel van der Lelie for providing the bacteria CH34; Takeharu Nagai for sharing the FRET backbone of YC 3.6; Chia-Lin Wu for gifs of fly strains Cha-gal4, R13F02-gal4, and TH-gal4.

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                              Keywords:

                              Blood lead level; Environmental Pb detection; biosensor; Fluorescence resonance energy transfer; Fluorescent biosensors; Genetically-encoded fluorescent protein biosensors; in-cell Pb biosensing; Heavy metal lead

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